Albumin variants

ABSTRACT

The present invention relates to variants of a parent albumin having altered plasma half-life compared with the parent albumin. The present invention also relates to fusion polypeptides and conjugates comprising said variant albumin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/863,780,filed Sep. 24, 2015, pending, which is a division of U.S. applicationSer. No. 14/262,244, filed Apr. 25, 2014, pending, which is a divisionof U.S. application Ser. No. 13/504,326 filed Apr. 26, 2012 (now U.S.Pat. No. 8,748,380), which is a 35 U.S.C. 371 national application ofPCT/EP2010/066572 filed Nov. 1, 2010, which claims priority or thebenefit under 35 U.S.C. 119 of European application nos. 10174162.7 and09174698.2 filed Aug. 26, 2010 and Oct. 30, 2009, respectively, and U.S.provisional application Nos. 61/348,001 and 61/327,171 filed May 25,2010 and Apr. 23, 2010, respectively, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to variants of albumin or fragmentsthereof or fusion polypeptides comprising variant albumin or fragmentsthereof having a change in half-life compared to the albumin, fragmentthereof or fusion polypeptide comprising albumin or a fragment thereof.

Description of the Related Art

Albumin is a protein naturally found in the blood plasma of mammalswhere it is the most abundant protein. It has important roles inmaintaining the desired osmotic pressure of the blood and also intransport of various substances in the blood stream.

Albumins have been characterized from many species including human, pig,mouse, rat, rabbit and goat and they share a high degree of sequence andstructural homology.

Albumin binds in vivo to its receptor, the neonatal Fc receptor (FcRn)“Brambell” and this interaction is known to be important for the plasmahalf-life of albumin. FcRn is a membrane bound protein, expressed inmany cell and tissue types. FcRn has been found to salvage albumin fromintracellular degradation (Roopenian D. C. and Akilesh, S. (2007), Nat.Rev. Immunol 7, 715-725.). FcRn is a bifunctional molecule thatcontributes to maintaining a high level of IgGs and albumin in serum inmammals such as human beings.

Whilst the FcRn-immunoglobulin (IgG) interaction has been characterizedin the prior art, the FcRn-albumin interaction is less wellcharacterized. The major FcRn binding site is localized within DIII(381-585). Andersen et al (2010). Clinical Biochemistry 43, 367-372.Data indicates that IgG and albumin bind non-cooperatively to distinctsites on FcRn (Andersen et al. (2006), Eur. J. Immunol 36, 3044-3051;Chaudhury et al. (2006), Biochemistry 45, 4983-4990.).

It is known that mouse FcRn binds IgG from mice and humans whereas humanFcRn appears to be more discriminating (Ober et al. (2001) Int. Immunol13, 1551-1559). Andersen et al. (2010). Journal of Biological Chemistry285(7):4826-36, describes the affinity of human and mouse FcRn for eachmouse and human albumin (all possible combinations). No binding ofalbumin from either species was observed at physiological pH to eitherreceptor. At acidic pH, a 100-fold difference in binding affinity wasobserved. In all cases, binding of albumin and IgG from either speciesto both receptors were additive.

Human serum albumin (HSA) has been well characterized as a polypeptideof 585 amino acids, the sequence of which can be found in Peters, T.,Jr. (1996) All about Albumin: Biochemistry, Genetics and Medical,Applications pp 10, Academic Press, Inc., Orlando (ISBN 0-12-552110-3).It has a characteristic binding to its receptor FcRn, where it binds atpH 6.0 but not at pH 7.4.

The plasma half-life of HSA has been found to be approximately 19 days.A natural variant having lower plasma half-life has been identified(Peach, R. J. and Brennan, S. O., (1991) Biochim Biophys Acta.1097:49-54) having the substitution D494N. This substitution generatedan N-glycosylation site in this variant, which is not present in thewild-type albumin. It is not known whether the glycosylation or theamino acid change is responsible for the change in plasma half-life.

Albumin has a long plasma half-life and because of this property it hasbeen suggested for use in drug delivery. Albumin has been conjugated topharmaceutically beneficial compounds (WO 2000/69902A), and it was foundthat the conjugate—maintained the long plasma half-life of albumin. Theresulting plasma half-life of the conjugate was generally considerablylonger than the plasma half-life of the beneficial therapeutic compoundalone.

Further, albumin has been fused to therapeutically beneficial peptides(WO 2001/79271 A and WO 2003/59934 A) with the typical result that thefusion has the activity of the therapeutically beneficial peptide and aconsiderably longer plasma half-life than the plasma half-life of thetherapeutically beneficial peptides alone.

Otagiri et al (2009), Biol. Pharm, Bull. 32(4), 527-534, discloses that77 albumin variant are know, of these 25 are found in domain III. Anatural variant lacking the last 175 amino acids at the carboxy terminihas been shown to have reduced half-life (Andersen et al (2010),Clinical Biochemistry 43, 367-372). Iwao et al. (2007) studied thehalf-life of naturally accuring human albumin variants using a mousemodel, and found that K541E and K560E had reduced half-life, E501K andE570K had increased half-life and K573E had almost no effect onhalf-life (Iwao, et. al. (2007) B.B.A. Proteins and Proteomics 1774,1582-1590).

Galliano et al (1993) Biochim. Biophys. Acta 1225, 27-32 discloses anatural variant E505K. Minchiotti et al. (1990) discloses a naturalvariant K536E. Minchiotti et al (1987) Biochim. Biophys. Acta 916,411-418 discloses a natural variant K574N. Takahashi et al (1987) Proc.Natl. Acad. Sci. USA 84, 4413-4417, discloses a natural variant D550G.Carlson et al (1992). Proc. Nat. Acad. Sci. USA 89, 8225-8229, disclosesa natural variant D550A.

Albumin has the ability to bind a number of ligands and these becomeassociated (associates) with albumin. This property has been utilized toextend the plasma half-life of drugs having the ability to noncovalentlybind to albumin. This can also be achieved by binding a pharmaceuticalbeneficial compound, which has little or no albumin binding properties,to a moiety having albumin binding properties. See review article andreference therein, Kratz (2008). Journal of Controlled Release 132,171-183.

Albumin is used in preparations of pharmaceutically beneficialcompounds, in which such a preparation maybe for example, but notlimited to, a nano particle or micro particle of albumin. In theseexamples the delivery of a pharmaceutically beneficial compound ormixture of compounds may benefit from alteration in the albuminsaffinity to its receptor where the beneficial compound has been shown toassociate with albumin for the means of delivery.

It is not clear what determines the plasma half-life of the formedassociates (for example but not limited to Levemir®, Kurtzhals P et al.Biochem. J. 1995; 312:725-731) conjugates or fusion polypeptides but itappears to be a result of the combination of the albumin and theselected pharmaceutically beneficial compound/polypeptide. It would bedesirable to be able to control the plasma half-life of given albuminconjugates, associates or albumin fusion polypeptides so that a longeror shorter plasma half-life can be achieved than given by the componentsof the association, conjugation or fusion, in order to be able to designa particular drug according to the particulars of the indicationintended to be treated.

Albumin is known to accumulate and be catabolised in tumours, it hasalso been shown to accumulate in inflamed joints of rheumatoid arthritissufferers. See review article and reference therein, Kratz (2008).Journal of Controlled Release 132, 171-183. It is envisaged that HSAvariants with increased affinity for FcRn would be advantageous for thedelivery of pharmaceutically beneficial compounds.

It may even be desirable to have variants of albumin that have little orno binding to FcRn in order to provide shorter half-lives or controlledserum pharmacokinetics as described by Kenanova et al (2009) J. Nucl.Med.; 50 (Supplement 2):1582).

SUMMARY OF THE INVENTION

The present invention provides variants of a parent albumin withimproved properties compared to its parent. In particular the inventionprovides variants of a parent albumin having altered plasma half-lifecompare to its parent.

The present invention relates to isolated variants of albumin orfragments thereof, or fusion polypeptides comprising variant albumin orfragments thereof, of a parent albumin, comprising an alteration at oneor more (several) positions corresponding to positions 417, 440, 464,490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510,535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579,580, 581, 582 and 584 of the mature polypeptide of SEQ ID NO: 2, whereinthe variant is not the variant consisting of SEQ ID NO: 2 with thesubstitution D494N, E501K, K541E, D550G,A, K573E or K574N.

The alteration at one or more position may independently be selectedamong substitutions, insertions and deletions, where substitution arepreferred.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to conjugates or associatescomprising the variant albumin or fragment thereof according to theinvention and a beneficial therapeutic moiety or to a fusion polypeptidecomprising a variant albumin or fragment thereof of the invention and afusion partner polypeptide.

The invention further relates to compositions comprising the variantalbumin, fragment thereof, fusion polypeptide comprising variant albuminor fragment thereof or conjugates comprising the variant albumin orfragment thereof, according to the invention or associates comprisingthe variant albumin or fragment thereof, according to the invention. Thecompositions are preferably pharmaceutical compositions.

The invention further relates to a pharmaceutical composition comprisinga variant albumin, fragment thereof, fusion polypeptide comprisingvariant albumin or fragment thereof or conjugates comprising the variantalbumin or fragment thereof, or associates comprising the variantalbumin or fragment thereof, wherein said variant albumin, fragmentthereof, fusion polypeptide comprising variant albumin or fragmentthereof or conjugates comprising the variant albumin or fragment orassociates of variant albumin or fragment thereof has altered plasmahalf-life compared to the corresponding plasma half-life of the HSA orfragment thereof, fusion polypeptide comprising HSA or fragment thereofor conjugates or associates of HSA or, fragment thereof, comprising HSAor fragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of the expression plasmid pDB4082.

FIG. 2 shows a restriction map of the expression plasmid pDB2305

FIG. 3 shows a restriction map of the expression plasmid pDB4005

FIG. 4 shows SPR sensorgrams 10 μM albumin injected over shFcRn HSA(JTA)=fatty acid free HSA obtained from Sigma-Aldrich (A3782), HSA(Novozymes)=Commercial Recombinant human serum albumin (RECOMBUMIN).

FIG. 5 shows ELISA binding of shFcRn-GST to human serum albumin (HSA)variants (100-0.045 μg/ml). Binding of WT, D494N, D494Q and D494A pH 6.0and pH 7.4. Binding of WT, D494N, D494N/T496A and T496A at pH 6.0 and pH7.4. Binding of WT, E495Q and E495A at pH 6.0 and pH 7.4.

FIG. 6 shows representative sensorgrams of binding of 0.2 μM of HSAvariants to immobilized shFcRn (˜4600 RU). WT, D494N, D494Q, D494A,D494N/T496A and T496A.

FIG. 7 shows representative sensorgrams of binding of 1 μM of HSAvariants to immobilized shFcRn (˜1400 RU). WT, D494N, D494Q, D494A,D494N/T496A and T496A.

FIG. 8 shows relative binding of the HSA variants compared to WT basedon two independent SPR experiments as shown (A) FIG. 6 and (B) FIG. 7.

FIG. 9 shows ELISA: (A) binding of shFcRn to albumins from human,donkey, bovine, sheep, goat and rabbit at pH 6.0. (B) binding of shFcRnto albumin from guinea pig, hamster, rat and chicken at pH 6.0. (C)binding of shFcRn to albumin from human, donkey, bovine, sheep, goat andrabbit at pH 7.4. (D) binding of shFcRn to albumin from guinea pig,hamster, rat and chicken at pH 7.4. (E) relative binding of thedifferent albumins. Relative binding of human albumin to shFcRn isdefined as 1.0. The ELISA values represent the mean of duplicates.

FIG. 10 shows SPR: Binding of shFcRn-GST to albumin from several speciesat pH 6.0 and pH 7.4. Representative sensorgrams showing binding of 5.0μM of albumin from different species; (A) human, (B) donkey, (C) bovine,(D) goat, (E) sheep, (F) rabbit, (G) dog, (H) guinea pig, (I) hamster,(J) rat, (K) mouse and (L) chicken. The albumin variants were injectedover immobilized GST-tagged shFcRn (˜2100 RU). Injections were performedat 25° C. at a rate of 40 μl/min.

FIG. 11 shows SPR sensorgrams of selected HSA mutants compared withwild-type HSA. 20 μM of (A) WT and P499A (B) WT and K500A, (C) WT andK536A, (D) WT and P537A and (E) WT and K538A and (F) WT and K537A wereinjected over immobilized shFcRn at pH 6.0 (˜1500 RU)

FIG. 12 shows SPR sensorgrams of HSA mutants compared with WT HSA. 10 μMof (A) WT and K573A (B) WT and K573C, (C) WT and K573F, (D) WT and K573Gand (E) WT and K573L and (F) WT and K573M, (G) WT and K573Q, (H) WT andK573R and (I) WT and K573T and (J) WT and K573V injected overimmobilized shFcRn at pH 5.5 and pH7.4. Injections were performed at 25°C. at a flow rate of 80 μl/min.

FIG. 13 shows SPR sensorgrams of HSA mutants compared with wild-typeHSA. 10 μM of (A) WT and K573D (B) WT and K573E, (C) WT and K573H, (D)WT and K5731 and (E) WT and K573N and (F) WT and K573P, (G) WT andK573S, (H) WT and K573* and (I) WT and K573W and (J) WT and K573Yinjected over immobilized shFcRn at pH 5.5 and pH7.4. Injections wereperformed at 25° C. at a flow rate of 80 μl/min.

FIG. 14 shows SPR sensorgrams of HSA mutants compared with wild-typeHSA. 20 μM of (A) WT and E492G+K538H+K541N+E542D (B) WT andE492T+N503K+K541A, (C) WT and E492P+N503K+K541G+E542P, (D) WT andE492H+E501P+N503H+E505D+T506S+T540S+K541E and (E) WT andA490D+E492T+V493L+E501P+N503D+A504E+E505K+T506F+K541D and (F) WT andE492G+V493P+K538H+K541N+E542D injected over immobilized shFcRn at pH6.0. Injections were performed at 25° C. at a flow rate of 80 μl/min.

FIG. 15 shows SPR sensorgrams of HSA mutants compared with wild-typeHSA. Twenty μM of (A) WT, (B) H440Q, (C) H464Q and (D) H535Q injectedover immobilized shFcRn at pH 6.0. Injections were performed at 25° C.at a flow rate of 80 μl/min.

FIG. 16 shows SPR sensorgrams of HSA mutant K500E compared withwild-type HSA. Ten μM of HSA mutant K500E injected over immobilizedshFcRn at pH 5.75. Injections were performed at 25° C. at a flow rate of30 μl/min.

FIG. 17 shows a restriction map of the expression plasmid pDB3017

FIG. 18 shows a restriction map of the expression plasmid pDB3021

FIG. 19 shows a restriction map of the expression plasmid pDB3056

FIG. 20 shows a restriction map of the expression plasmid pDB3165

FIG. 21 shows a restriction map of the expression plasmid pDB4172

FIG. 22 shows a restriction map of the expression plasmid pDB4267

FIG. 23 shows a restriction map of the expression plasmid pDB4285

FIG. 24 shows a GP-HPLC chromatogram of WT HSA and mutant K573P HRPconjugates for shFcRn analysis. Injections of 25 μL were made onto a TSKG3000SWXL column (Tosoh Bioscience) as described in materials andmethods.

FIG. 25 shows SDS PAGE separation followed by both visual (A) andultraviolet (B) detection of the Fluorescein conjugated albumin.HSA::FSM (Lane 1), K573P::F5M (Lane 2) and rHA standard (Lane 3).

FIG. 26 shows shFcRn binding properties of HSA variants. 10 μM of WT rHAand E492T(A), WT rHA and D494N/E495Q/T496A(B), WT rHA and N503D(C), WTrHA and N503K(D), WT rHA and E492T/N503D(E), WT rHA and E495Q/T496A(F),WT rHA and K538H(G), WT rHA and E492D(H) injected over immobilisedshFcRn at pH5.5 FIG. 27 shows shFcRn binding properties of HSA variants.10 μM of WT rHA and K541A(I) and WT rHA and K541N(J) were injected overimmobilised shFcRn at pH5.5.

FIG. 28 shows competitive binding of K573A and K573P measured byinjecting shFcRn (100 nM) alone or pre-incubated with different amountsof HSA K573A and K573P over immobilized HSA (˜2500 RU) at pH6.0 FIG. 29shows competitive binding of HSA-FLAG variants measured by injectingshFcRn (100 nM) alone or together with different amounts of HSA-FLAGvariants over immobilized HSA (˜2500 RU) at pH6.0.

FIG. 30 shows competitive binding of HSA-IL1Ra variants measured byinjecting shFcRn (100 nM) alone or together with different amounts ofHSA-IL1Ra variants over immobilized HSA (˜2500 RU) at pH6.0

FIG. 31 shows competitive binding of scFv-fused HSA variants measured byinjecting shFcRn (100 nM) alone or together with different amounts of(A) scFv-HSA-FLAG variants or (B) HSA-scFv-FLAG variants overimmobilized HSA (˜2500 RU) at pH6.0.

FIG. 32 shows binding of HSA, single, double and triple mutant variantsto shFcRn. Samples of 10 μM of each HSA variant were injected overimmobilized shFcRn at pH 5.5 or pH 7.4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of albumin orfragments thereof, or fusion polypeptides comprising variant albumin orfragments thereof, of a parent albumin, comprising an alteration at oneor more (several) positions corresponding to positions 417, 440, 464,490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510,535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579,580, 581, 582 and 584 of the mature polypeptide of SEQ ID NO: 2, whereinthe variant is not the variant consisting of SEQ ID NO: 2 with thesubstitution D494N, E501K, K541E, D550G,A, K573E or K574N.

The alteration at one or more position may independently be selectedamong substitutions, insertions and deletions, where substitution arepreferred.

Definitions

Variant: The term “variant” means a polypeptide derived from a parentalbumin by one or more alteration(s), i.e., a substitution, insertion,and/or deletion, at one or more (several) positions. A substitutionmeans a replacement of an amino acid occupying a position with adifferent amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding 1 or more,preferably 1-3 amino acids immediately adjacent to an amino acidoccupying a position.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Wild-Type Albumin: The term “wild-type” (WT) albumin means albuminhaving the same amino acid sequence as naturally found in an animal orin a human being.

Parent or Parent albumin The term “parent” or “parent albumin” means analbumin to which an alteration is made by the hand of man to produce thealbumin variants of the present invention. The parent may be a naturallyoccurring (wild-type) polypeptide or an allele thereof, or even avariant thereof.

FcRn and shFcRn: The term “FcRn” means the human neonatal Fc receptor(FcRn). shFcRn is a soluble recombinant form of FcRn.

smFcRn: The term “smFcRn” is a soluble recombinant form of the mouseneonatal Fc Receptor.

Isolated variant: The term “isolated variant” means a variant that ismodified by the hand of man and separated completely or partially fromat least one component with which it naturally occurs. In one aspect,the variant is at least 1% pure, e.g., at least 5% pure, at least 10%pure, at least 20% pure, at least 40% pure, at least 60% pure, at least80% pure, and at least 90% pure, as determined by SDS-PAGE or GP-HPLC.

Substantially pure variant: The term “substantially pure variant” meansa preparation that contains at most 10%, at most 8%, at most 6%, at most5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% byweight of other polypeptide material with which it is natively orrecombinantly associated. Preferably, the variant is at least 92% pure,e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100%pure by weight of the total polypeptide material present in thepreparation. The variants of the present invention are preferably in asubstantially pure form. This can be accomplished, for example, bypreparing the variant by well known recombinant methods and bypurification methods.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 585 of SEQ ID NO: 2, with the inclusionof any post-translational modifications.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature albuminpolypeptide. In one aspect, the mature polypeptide coding sequence isnucleotides 1 to 1758 of SEQ ID NO: 1.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labelled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofan albumin and/or an internal region of albumin that has retained theability to bind to FcRn. Fragments may consist of one uninterruptedsequence derived from HSA or it may comprise two or more sequencesderived from HSA. The fragments according to the invention have a sizeof more than approximately 20 amino acid residues, preferably more than30 amino acid residues, more preferred more than 40 amino acid residues,more preferred more than 50 amino acid residues, more preferred morethan 75 amino acid residues, more preferred more than 100 amino acidresidues, more preferred more than 200 amino acid residues, morepreferred more than 300 amino acid residues, even more preferred morethan 400 amino acid residues and most preferred more than 500 amino acidresidues.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of its translatedpolypeptide product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a variant ofthe present invention. Each control sequence may be native or foreign tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences within the coding region of the polynucleotideencoding a variant.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of the variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to additional nucleotides that provide for itsexpression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Plasma half-life: Plasma half-life is ideally determined in vivo insuitable individuals. However, since it is time consuming and expensiveand there inevitable are ethical concerns connected with doingexperiments in animals or man it is desirable to use an in vitro assayfor determining whether plasma half-life is extended or reduced. It isknown that the binding of albumin to its receptor FcRn is important forplasma half-life and the correlation between receptor binding and plasmahalf-life is that a higher affinity of albumin to its receptor leads tolonger plasma half-life. Thus for the present invention a higheraffinity of albumin to FcRn is considered indicative of an increasedplasma half-life and a lower affinity of albumin to its receptor isconsidered indicative of a reduced plasma half-life.

In this application and claims the binding of albumin to its receptorFcRn is described using the term affinity and the expressions “stronger”or “weaker”. Thus, it should be understood that a molecule having ahigher affinity to FcRn than HSA is considered to bind stronger to FcRnthan HSA and a molecule having a lower affinity to FcRn than HSA isconsidered to bind weaker to FcRn than HSA.

The terms “longer plasma half-life” or “shorter plasma half-life” andsimilar expressions are understood to be in relationship to thecorresponding parent albumin molecule. Thus, a longer plasma half-lifewith respect to a variant albumin of the invention means that thevariant has longer plasma half-life than the corresponding albuminhaving the same sequences except for the alteration(s) in positionscorresponding to 417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500,501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550,573, 574, 575, 577, 578, 579, 580, 581, 582 and 584 in SEQ ID NO: 2.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another albumin. The amino acid sequence of another albuminis aligned with the mature polypeptide disclosed in SEQ ID NO: 2, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the mature polypeptide disclosed in SEQ ID NO:2 is determined using the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleprogram of the EMBOSS package (EMBOSS: The European Molecular BiologyOpen Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),preferably version 3.0.0 or later.

Identification of the corresponding amino acid residue in anotheralbumin can be confirmed by an alignment of multiple polypeptidesequences using “ClustalW” (Larkin et al., 2007, Bioinformatics 23:2947-2948).

When the other polypeptide (or protein) has diverged from the maturepolypeptide of SEQ ID NO: 2 such that traditional sequence-basedcomparison fails to detect their relationship (Lindahl and Elofsson,2000, J. Mol. Biol. 295: 613-615), other pairwise sequence comparisonalgorithms can be used. Greater sensitivity in sequence-based searchingcan be attained using search programs that utilize probabilisticrepresentations of polypeptide families (profiles) to search databases.For example, the PSI-BLAST program generates profiles through aniterative database search process and is capable of detecting remotehomologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Evengreater sensitivity can be achieved if the family or superfamily for thepolypeptide has one or more representatives in the protein structuredatabases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilizeinformation from a variety of sources (PSI-BLAST, secondary structureprediction, structural alignment profiles, and solvation potentials) asinputs to a neural network that predicts the structural fold for a querysequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol.313: 903-919, can be used to align a sequence of unknown structurewithin the superfamily models present in the SCOP database. Thesealignments can in turn be used to generate homology models for thepolypeptide, and such models can be assessed for accuracy using avariety of tools developed for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementations of thesealgorithms can additionally be utilized to query structure databaseswith a structure of interest in order to discover possible structuralhomologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the albumin variants of the present invention, thenomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, forexample the substitution of threonine with alanine at position 226 isdesignated as “Thr226Ala” or “T226A”. Multiple mutations are separatedby addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”,representing substitutions at positions 205 and 411 of glycine (G) witharginine (R) and serine (S) with phenylalanine (F), respectively. TheFigures also use (“/”), e.g., “E492T/N503D” this should be viewed asinterchangeable with (“+”).

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position*. Accordingly, the deletion of glycine at position195 is designated as “Gly195*” or “G195*”. Multiple deletions areseparated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of tyrosine and glutamic acid for arginine and glycine atpositions 170 and 195, respectively.

Different Substitutions.

Where different substitutions can be introduced at a position, thedifferent substitutions are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine with tyrosine or glutamic acid atposition 170. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”. Parent Albumin

Albumins are proteins and constitute the most abundant protein in plasmain mammals and albumins from a long number of mammals have beencharacterized by biochemical methods and/or by sequence information.Several albumins, e.g., human serum albumin (HSA), have also beencharacterized crystallographically and the structure determined.

HSA is a preferred albumin according to the invention and is a proteinconsisting of 585 amino acid residues and has a molecular weight of 67kDa. In its natural form it is not glycosylated. The amino acid sequenceof HSA is shown in SEQ ID NO: 2. The skilled person will appreciate thatnatural alleles may exist having essentially the same properties as HSAbut having one or more amino acid changes compared to SEQ ID NO: 2, andthe inventors also contemplate the use of such natural alleles as parentalbumin according to the invention.

Albumins have generally a long plasma half-life of approximately 20 daysor longer, e.g., HSA has a plasma half-life of 19 days. It is known thatthe long plasma half-life of HSA is mediated via interaction with itsreceptor FcRn, however, an understanding or knowledge of the exactmechanism behind the long half-life of HSA is not essential for thepresent invention.

According to the invention the term “albumin” means a protein having thesame, or very similar three dimensional structure as HSA and having along plasma half-life. As examples of albumin proteins according to theinvention can be mentioned human serum albumin, primate serum albumin,(such as chimpanzee serum albumin, gorilla serum albumin), rodent serumalbumin (such as hamster serum albumin, guinea pig serum albumin, mousealbumin and rat serum albumin), bovine serum albumin, equine serumalbumin, donkey serum albumin, rabbit serum albumin, goat serum albumin,sheep serum albumin, dog serum albumin, chicken serum albumin and pigserum albumin. HSA as disclosed in SEQ ID NO: 2 or any naturallyoccurring allele thereof, is the preferred albumin according to theinvention.

The parent albumin, a fragment thereof, or albumin part of a fusionpolypeptide comprising albumin or a fragment thereof according to theinvention has generally a sequence identity to the sequence of HSA shownin SEQ ID NO: 2 of at least 60%, preferably at least 70%, preferably atleast 80%, preferably at least 85%, preferably at least 86%, preferablyat least 87%, preferably at least 88%, preferably at least 89%,preferably at least 90%, preferably at least 91%, preferably at least92%, preferably at least 93%, preferably at least 94%, preferably atleast 95%, more preferred at least 96%, more preferred at least 97%,more preferred at least 98% and most preferred at least 99%.

The parent preferably comprises or consists of the amino acid sequenceof SEQ ID NO: 2. In another aspect, the parent comprises or consists ofthe mature polypeptide of SEQ ID NO: 2.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2.

In a second aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii)the full-length complementary strand of (i) or (ii) (J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labelled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labelled nucleotide probe correspondingto the polynucleotide shown in SEQ ID NO: 1, its complementary strand,or a subsequence thereof, under low to very high stringency conditions.Molecules to which the probe hybridizes can be detected using, forexample, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 1 to 1785 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2 or a fragment thereof. In another aspect, the nucleic acid probeis SEQ ID NO: 1.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), 50° C.(low stringency), 55° C. (medium stringency), 60° C. (medium-highstringency), 65° C. (high stringency), or 70° C. (very high stringency).

For short probes that are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In a third aspect, the parent is encoded by a polynucleotide with asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which encodes apolypeptide which is able to function as an albumin. In an embodiment,the parent is encoded by a polynucleotide comprising or consisting ofSEQ ID NO: 1.

Preparation of Variants

In a further aspect the invention relates to a method for preparing avariant albumin, fragment thereof, or fusion polypeptide comprisingvariant albumin or a fragment thereof comprising the steps of:

-   -   a. Identifying one or more amino acid residue positions being        important for the binding of albumin to FcRn, in an albumin or a        fragment thereof or the albumin part of a fusion polypeptide        comprising albumin or a fragment thereof;    -   b. Providing a nucleic acid encoding said albumin, the fragment        thereof or the albumin part of a fusion polypeptide comprising        albumin or the fragment thereof;    -   c. Modifying the nucleic acid provided in b., so that the one or        more (several) amino acid residue located at the positions        identified in a., are deleted or substituted or inserted with a        different amino acid;    -   d. Expressing the modified nucleic acid in a suitable host cell;        and    -   e. Recovering the variant albumin, the fragment thereof or the        fusion polypeptide comprising variant albumin or the fragment        thereof.

The identification of one or more amino acid residue positions beingimportant for the binding of albumin to FcRn, in albumin, fragmentthereof or the albumin part of a fusion polypeptide can be done inseveral ways including, but not limited to, random mutagenesis followedby analysis of the generated mutants and comparison with the non-mutatedparent molecule, and identification based on structural considerationsoptionally followed by generation of variants having the identifiedalterations and comparison with the non-mutated patent molecule.

A preferred method for identification of one or more amino acid residuepositions to be changed to in order to prepare a variant HSA having analtered binding to FcRn compared with natural HSA, comprises thefollowing steps:

-   -   i) Identifying a non-human albumin having a different binding        property to FcRn;    -   ii) Identifying the amino acid residues of the human serum        albumin interacting with FcRn;    -   iii) Comparing the primary and/or the tertiary structure of the        identified non-human albumin and human serum albumin with        respect to the amino acid residues identified in step ii) and        identifying the amino acid residues that differ between said        non-human albumin and human serum albumin as being responsible        for the observed binding difference; and    -   iv) Optionally preparing variants of HSA at the positions        identified in step iii) and confirming that the prepared        variants have altered binding to FcRn compared with HSA.

Step i) above may be done using the SPR assay described below. However,the skilled person will appreciate that other methods may be used toidentify non-human albumins having different binding properties to FcRnthan HSA, and that the method is not dependent on how the non-humanalbumin, having different binding properties to FcRn, has beenidentified.

In one preferred embodiment the identified non-human albumin has astronger binding to FcRn than HSA. Examples of non-human albumins havingstronger binding to FcRn than HSA include donkey serum albumin, rabbitserum albumin, dog serum albumin, hamster serum albumin, guinea pigserum albumin, mouse serum albumin and rat serum albumin. Step ii) maybe accomplished by considering the structure of FcRn, HSA and thebinding complex of these two. In the absence of an available structureof the binding complex it is possible to use a model where the HSAstructure is docked into the structure of the FcRn structure and therebyidentify amino acid residues of HSA interacting with FcRn.

In another preferred embodiment the identified non-human albumin has aweaker binding to FcRn than HSA. Examples of non-human albumins havingweaker binding to FcRn than HSA include bovine serum albumin, goat serumalbumin, sheep serum albumin and chicken serum albumin. Step ii) may beaccomplished by considering the structure of FcRn, HSA and the bindingcomplex of these two. In absence of an available structure of thebinding complex it is possible to use a model where the HSA structure isdocked into the structure of the FcRn structure and thereby identifyresidues of HSA interacting with FcRn.

In this invention and claims, an amino acid residues of HSA interactingwith FcRn is considered any amino acid residues of HSA being locatedless than 10 Å from an amino acid in the FcRn or any amino acid residuethat is involved in a hydrogen bond, a salt bridge or a polar ornonpolar interaction with an amino acid residue that is located lessthan 10 Å from an amino acid in the FcRn. Preferably the amino acid inHSA residues are located less than 10 Å from amino acids in the FcRn,more preferred less than 6 Å from amino acids in the FcRn and mostpreferred less than 3 Å from amino acids in the FcRn.

Step iii) and iv) can be done using techniques well known to the skilledperson.

The present invention also relates to methods for obtaining a variantalbumin or fragments thereof, or fusion polypeptides comprising thevariant albumin or fragments thereof, or associates of variant albuminor fragment thereof comprising: (a) introducing into a parent albumin orfragments thereof, or fusion polypeptides comprising the parent albuminor fragments thereof an alteration at one or more (several) positionscorresponding to positions 417, 440, 464, 490, 492, 493, 494, 495, 496,499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541,542, 550, 573, 574, 575, 577, 578, 579, 580, 581, 582 and 584 of themature polypeptide of SEQ ID NO: 2; and (b) recovering the variantalbumin or fragments thereof, or fusion polypeptides comprising thevariant albumin or fragments thereof.

The variants can be prepared by those skilled persons using anymutagenesis procedure known in the art, such as site-directedmutagenesis, synthetic gene construction, semi-synthetic geneconstruction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (several)mutations are created at one or more defined sites in a polynucleotideencoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests at theplasmid and the oligonucleotide is the same, permitting ligation of theplasmid and insert to one another. See, e.g., Scherer and Davis, 1979,Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990,Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein olgionucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide sub sequences may then be shuffled.

Variants

The present invention also provides variant albumins or fragmentsthereof, or fusion polypeptides comprising the variant albumin orfragments thereof, of a parent albumin, comprising an alteration at oneor more (several) positions corresponding to positions 417, 440, 464,490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510,535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579,580, 581, 582 and 584 in SEQ ID NO: 2, wherein each alteration isindependently a substitution, insertion or deletion with the provisionthat the and the variant is not SEQ ID NO: 2 having the substitutionD494N, E501K, K541E, D550G,A, K573E or K574N.

The variant albumin, a fragment thereof, or albumin part of a fusionpolypeptide comprising variant albumin or a fragment thereof accordingto the invention has generally a sequence identity the sequence of HSAshown in SEQ ID NO: 2 of at least 60%, preferably at least 70%,preferably at least 80%, preferably at least 85%, preferably at least90%, more preferred at least 95%, more preferred at least 96%, morepreferred at least 97%, more preferred at least 98% and most preferredat least 99%.

In one aspect, the number of alterations in the variants of the presentinvention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 alterations.

The variant albumin, a fragment thereof or fusion polypeptide comprisingthe variant albumin or fragment thereof has altered plasma half-lifecompared with the corresponding parent albumin, fragment thereof, orfusion polypeptide comprising the variant albumin or fragment thereof.

In a particular preferred embodiment the parent albumin is HSA and thevariant albumin, a fragment thereof or fusion polypeptide comprising thevariant albumin or fragment thereof has altered plasma half-lifecompared with the HSA, the corresponding fragment or fusion polypeptidecomprising HSA or fragment thereof.

The correlation between binding of albumin to its receptor and plasmahalf-life has been realized by the present inventors based on thenatural occurring allele of HSA D494N. The inventors have analyzed thisallele and found that it has a lower affinity to its receptor FcRn.

Further, it has been disclosed that a transgenic mouse having thenatural mouse FcRn replaced with human FcRn has a higher serum albuminlevel than normal mouse; see (J Exp Med. (2003) 197(3):315-22). Theinventors have discovered that human FcRn has a higher affinity to mouseserum albumin than mouse FcRn has to mouse serum albumin and, therefore,the observed increase in serum albumin in the transgenic micecorresponds with a higher affinity between serum albumin and itsreceptor, confirming the correlation between albumin binding to FcRn andplasma half-life. In addition, variants of albumin that have little orno binding to FcRn have been shown to have reduced half-life in a mousemodel, Kenanova et al (2009) J. Nucl. Med.; 50 (Supplement 2): 1582).

One way to determine whether the affinity of a variant albumin to FcRnis higher or lower than the parent albumin is to use the Surface PlasmonResonance assay (SPR) as described below. The skilled person willunderstand that other methods might be useful to determine whether theaffinity of a variant albumin to FcRn is higher or lower than theaffinity of the parent albumin to FcRn, e.g., determination andcomparison of the binding constants KD. Thus, according to the inventionvariant albumins having a KD that is lower than the KD for natural HSAis considered to have a higher plasma half-life than HSA and variantalbumins having a KD that is higher than the KD for natural HSA isconsidered to have a lower plasma half-life than HSA.

The variants of albumin or fragments thereof or fusion polypeptidescomprising albumin or fragments thereof comprise one or morealterations, such as substitutions, deletions or insertions at one ormore (several) positions corresponding to the positions in HSA selectedfrom the group consisting of 417, 440, 464, 490, 492, 493, 494, 495,496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540,541, 542, 550, 573, 574, 575, 577, 578, 579, 580, 581, 582 and 584. Thesubstitution may be any substitution where the amino acid in the naturalalbumin sequence is substituted with a different amino acid selectedamong the remaining 19 natural occurring amino acids.

In one aspect, a variant comprises an alteration at one or more(several) positions corresponding to positions 417, 440, 464, 490, 492,493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536,537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579, 580, 581,582 and 584 in SEQ ID NO: 2. In another aspect, a variant comprises analteration at two positions corresponding to any of 417, 440, 464, 490,492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535,536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577, 578, 579, 580,581, 582 and 584 in SEQ ID NO: 2. In another aspect, a variant comprisesan alteration at three positions corresponding to any of positions 417,440, 464, 490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504, 505,506, 510, 535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575, 577,578, 579, 580, 581, 582 and 584 in SEQ ID NO: 2. In another aspect, avariant comprises an alteration at each position corresponding topositions 417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500, 501,503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550, 573,574, 575, 577, 578, 579, 580, 581, 582 and 584 in SEQ ID NO: 2.

In another aspect, the variant comprises the substitution Q417A,H of themature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution H440Q of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution H464Q ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution A490D of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution E492G,T,P,H of the mature polypeptide of SEQ ID NO: 2. In another aspect, thevariant comprises the substitution V493P,L of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises the substitutionD494N,Q,A,E,P of the mature polypeptide of SEQ ID NO: 2. In anotheraspect, the variant comprises the substitution E495Q,A of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the variant comprisesthe substitution T496A of the mature polypeptide of SEQ ID NO: 2. Inanother aspect, the variant comprises the substitution P499A of themature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution K500E,G,D,A,S,C,P,H,F,N,W,T,M,Y,V,Q,L,I,R ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution E501A,P,Q of the mature polypeptide of SEQ IDNO: 2. In another aspect, the variant comprises the substitutionN503K,D,H of the mature polypeptide of SEQ ID NO: 2. In another aspect,the variant comprises the substitution A504E of the mature polypeptideof SEQ ID NO: 2. In another aspect, the variant comprises thesubstitution E505K, D of the mature polypeptide of SEQ ID NO: 2. Inanother aspect, the variant comprises the substitution T506F, S of themature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution H510Q of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution H535Q ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution K536A of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution P537A ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution K538A,H of the mature polypeptide of SEQ IDNO: 2. In another aspect, the variant comprises the substitution T540Sof the mature polypeptide of SEQ ID NO: 2. In another aspect, thevariant comprises the substitution K541A,D,G,N,E of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the variant comprisesthe substitution E542P,D of the mature polypeptide of SEQ ID NO: 2. Inanother aspect, the variant comprises the substitution D550N of themature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution K573Y,W,P,H,F,V,I,T,N,S,G,M,C,A,E,Q,R,L,D ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution K574N of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution Q580K ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution L575F of the mature polypeptide of SEQ ID NO:2. In another aspect, the variant comprises the substitution A577T,E ofthe mature polypeptide of SEQ ID NO: 2. In another aspect, the variantcomprises the substitution A578R,S of the mature polypeptide of SEQ IDNO: 2. In another aspect, the variant comprises the substitution S579C,Tof the mature polypeptide of SEQ ID NO: 2. In another aspect, thevariant comprises the substitution Q580K of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises the substitutionA581D of the mature polypeptide of SEQ ID NO: 2. In another aspect, thevariant comprises the substitution A582T of the mature polypeptide ofSEQ ID NO: 2. In another aspect, the variant comprises the substitutionG584A of the mature polypeptide of SEQ ID NO: 2.

In one aspect, the variant comprises an alteration at a positioncorresponding to position 417. In another aspect, the amino acid at aposition corresponding to position 417 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala or His. In another aspect,the variant comprises the substitution Q417A, H of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 440. In another aspect, the amino acid at aposition corresponding to position 440 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution H440Q of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 464. In another aspect, the amino acid at aposition corresponding to position 464 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution H464Q of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 490 In another aspect, the amino acid at aposition corresponding to position 490 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In another aspect, the variant comprises thesubstitution A490G of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 492. In another aspect, the amino acid at aposition corresponding to position 492 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Gly. In another aspect, thevariant comprises the substitution E492G of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 493. In another aspect, the amino acid at aposition corresponding to position 493 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Pro. In another aspect, thevariant comprises the substitution V493P of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 494. In another aspect, the amino acid at aposition corresponding to position 494 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asn, Gln or Ala. In anotheraspect, the variant comprises the substitution D494N,Q, A of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 495. In another aspect, the amino acid at aposition corresponding to position 495 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Gln or Ala. In another aspect,the variant comprises the substitution E495Q or A of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 496. In another aspect, the amino acid at aposition corresponding to position 496 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution T496A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 499. In another aspect, the amino acid at aposition corresponding to position 499 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution P499A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 500. In another aspect, the amino acid at aposition corresponding to position 500 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitutionK500E,G,D,A,S,C,P,H,F,N,W,T,M,Y,V,Q,L,I,R of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 501. In another aspect, the amino acid at aposition corresponding to position 501 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala or Gln to reduce affinity andPro to increase affinity. In another aspect, the variant comprises thesubstitution E501A, Q, P of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 503. In another aspect, the amino acid at aposition corresponding to position 503 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asp or Lys or His. In anotheraspect, the variant comprises the substitution N503D, K, H of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 504. In another aspect, the amino acid at aposition corresponding to position 504 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In another aspect, the variant comprises thesubstitution A504 of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 505. In another aspect, the amino acid at aposition corresponding to position 505 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In another aspect, the variant comprises thesubstitution E505D of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 506. In another aspect, the amino acid at aposition corresponding to position 506 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In another aspect, the variant comprises thesubstitution T506S,F of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 510. In another aspect, the amino acid at aposition corresponding to position 510 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Gin. In another aspect, thevariant comprises the substitution H510Q of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 535. In another aspect, the amino acid at aposition corresponding to position 535 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Gin. In another aspect, thevariant comprises the substitution H535Q of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 536. In another aspect, the amino acid at aposition corresponding to position 536 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution K536A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 537. In another aspect, the amino acid at aposition corresponding to position 537 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution P537A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 538. In another aspect, the amino acid at aposition corresponding to position 538 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution K538H, A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 540. In another aspect, the amino acid at aposition corresponding to position 540 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val. In another aspect, the variant comprises thesubstitution T540S of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 541. In another aspect, the amino acid at aposition corresponding to position 541 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Gly, Asp or Ala. In anotheraspect, the variant comprises the substitution K541G, D A, N of themature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 542. In another aspect, the amino acid at aposition corresponding to position 542 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asp or Pro. In another aspect,the variant comprises the substitution E542D, P of the maturepolypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 550. In another aspect, the amino acid at aposition corresponding to position 550 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asn to reduce affinity,preferably with Glu to increase affinity.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 573. In another aspect, the amino acid at aposition corresponding to position 573 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Tyr, Trp, Pro, His. Phe, Val,Ile, Thr, Asn, Ser, Gly, Met, Cys, Ala, Glu, Gin, Arg, Leu, Asp. Inanother aspect, the variant comprises the substitutionK573Y,W,P,H,F,V,I,T,N,S,G,M,C,A,E,Q,R,L,D of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 574. In another aspect, the amino acid at aposition corresponding to position 574 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asn. In another aspect, thevariant comprises the substitution K574N of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 575. In another aspect, the amino acid at aposition corresponding to position 575 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Phe. In another aspect, thevariant comprises the substitution L575F of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 577. In another aspect, the amino acid at aposition corresponding to position 577 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Thr or Glu. In another aspect,the variant comprises the substitution A577TE of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 578. In another aspect, the amino acid at aposition corresponding to position 578 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Arg or Ser. In another aspect,the variant comprises the substitution A578R,S of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 579. In another aspect, the amino acid at aposition corresponding to position 579 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Cys or Thr. In another aspect,the variant comprises the substitution S579C,T of the mature polypeptideof SEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 580. In another aspect, the amino acid at aposition corresponding to position 580 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Lys. In another aspect, thevariant comprises the substitution Q580K of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 581. In another aspect, the amino acid at aposition corresponding to position 581 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Asp. In another aspect, thevariant comprises the substitution A581D of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 582. In another aspect, the amino acid at aposition corresponding to position 582 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Thr. In another aspect, thevariant comprises the substitution A582T of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at a positioncorresponding to position 584. In another aspect, the amino acid at aposition corresponding to position 584 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Ala. In another aspect, thevariant comprises the substitution G584A of the mature polypeptide ofSEQ ID NO: 2.

In another aspect, the variant comprises an alteration at positionscorresponding to positions 494 and 496 in SEQ ID NO: 2, such as thosedescribed above.

In another aspect, the variant comprises alterations at positionscorresponding to positions 492 and 493 in SEQ ID NO: 2, such as thosedescribed above.

In another aspect, the variant comprises alterations at positionscorresponding to positions 494 and 417 in SEQ ID NO: 2, such as thosedescribed above.

In another aspect, the variant comprises alterations at positionscorresponding to positions 492 and 503 in SEQ ID NO: 2, such as thosedescribed above.

In another aspect, the variant comprises alterations at positionscorresponding to positions 492 and 573 in SEQ ID NO: 2, such as thosedescribed above.

In another aspect, the variant comprises alterations at positionscorresponding to positions 492, 503, and 573 in SEQ ID NO: 2, such asthose described above.

In one embodiment the variant albumin or fragments thereof, or fusionpolypeptides comprising the variant albumin or fragments thereofaccording to the invention contains one substitution at a positioncorresponding to a position in HSA selected from the group consisting of417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500, 501, 503, 504,505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550, 573, 574, 575,577, 578, 579, 580, 581, 582 and 584 in SEQ ID NO: 2 provided that thevariant albumin is not the variant consisting of SEQ ID NO: 2 with thesubstitution D494N, E501K, K541E, D550G,A, K573E or K574N. The variantalbumin, fragment thereof or fusion polypeptides comprising variantalbumin or a fragment thereof according to the invention may compriseadditional substitutions, insertions or deletions at one or more(several) positions corresponding to other positions in HSA.

In another embodiment the variant albumin or fragments thereof, orfusion polypeptides comprising variant albumin or fragments thereofaccording to the invention contains two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen fifteen, sixteen,seventeen, eighteen, nineteen twenty or even more substitutions atpositions corresponding to positions in HSA selected from the groupconsisting of 417, 440, 464, 490, 492, 493, 494, 495, 496, 499, 500,501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542, 550,573, 574, 575, 577, 578, 579, 580, 581, 582 and 584 of SEQ ID NO: 2. Thevariant albumin or fragments thereof, or fusion polypeptides comprisingvariant albumin or fragments thereof according to the invention maycomprise additional substitutions, insertions or deletions at positionscorresponding to other positions in HSA.

In a further embodiment the variants of albumin or fragments thereof, orfusion polypeptides comprising variant albumin or a fragment thereofaccording to the invention have a plasma half-life that is longer thanthe plasma half-life of the parent albumin fragment thereof or fusionpolypeptide comprising the parent albumin or a fragment thereof.Examples according to this embodiment include variants of albumin orfragments thereof, or fusion polypeptides comprising variant albumin ora fragment thereof comprising a substitution in the positioncorresponding to 492, 503, 542, 550, 573, 574, 580, 581, 582 or 584 inSEQ ID NO: 2. Preferred substitutions according to this embodiment ofthe invention include the substitution of the amino acid residue in theposition corresponding to 492 in SEQ ID NO: 2 with a G residue,substitution of the amino acid residue in the position corresponding to503 in SEQ ID NO: 2 with a H or a K residue, substitution of the aminoacid residue in the position corresponding to 550 in SEQ ID NO: 2 withan E residue, the substitution of the amino acid residue in a positioncorresponding to 573 in SEQ ID NO: 2 with anY,W,P,H,F,V,I,T,N,S,G,M,C,A,E,Q,R,L or a D, the substitution of theamino acid residue in a position corresponding to 574 in SEQ ID NO: 2with an N residue, or the substitution of the amino acid residue in theposition corresponding to 580 in SEQ ID NO: 2 with an K residue. Otherpreferred variants have a substitution in the position corresponding to492 in SEQ ID NO: 2 with a G residue and a substitution in the positioncorresponding to 573 in SEQ ID NO: 2 with an A or a P residue. Otherpreferred variant has a number of substitutions corresponding toposition 492 in SEQ ID NO: 2 with an H residue in position 503 in SEQ IDNO: 2.

Other preferred variants have a substitution in the positioncorresponding to 492 in SEQ ID NO: 2 with a G residue and a substitutionin the position corresponding to position 503 in SEQ ID NO: 2corresponding to a H or a K and a substitution in position 573 in SEQ IDNO: 2 with an A or a P residue.

In a further embodiment the variants of albumin or fragments thereof, orfusion polypeptides comprising variant albumin or fragments thereofaccording to the invention have a plasma half-life that is shorter thanthe plasma half-life of the parent albumin fragment thereof or fusionpolypeptide comprising the parent albumin or a fragment thereof.Examples according to this embodiment include variants of albumin orfragments thereof, or fusion polypeptides comprising variant albumin ora fragment thereof comprising a substitution in the positioncorresponding to 417, 440, 494, 495, 496, 499, 500, 501, 536, 537, 538,541, 494+496 or 492+493 in SEQ ID NO: 2. Preferred substitutions includethe substitutions corresponding to Q417A, H440Q, D494E+Q417H, D494N,Q,A,E495Q,A, T496A, D494N+T496A or, P499A, K500A, E501A, E501Q, K536A,P537A, K538A, K541G, K541A K541D or D550N in SEQ ID NO: 2.

In another embodiment of the invention the variants of albumin orfragments thereof, or fusion polypeptides comprising variant albumin ora fragment thereof according to the invention have lost their ability tobind FcRn. In this connection variants of albumin or fragments thereof,or fusion polypeptides comprising variant albumin or fragments thereofis considered to have lost the ability to bind FcRn if the measuredresonance units for the variant in the SPR assay described below is lessthan 10% of the measured resonance units for the corresponding parentalbumin or fragment thereof. Examples according to this embodimentinclude variants of albumin or fragments thereof, or fusion polypeptidescomprising variant albumin or fragments thereof comprising asubstitution at a position corresponding to 464, 500, 510 or 535 in SEQID NO: 2. Preferred substitutions include the substitutionscorresponding to H464Q, K500A,P,C,S,A,D.G H510Q or H535Q in SEQ ID NO:2.

In addition to the one or more substitutions at one or more positionscorresponding to positions 417, 464, 490, 492, 493, 494, 495, 496, 499,500, 501, 503, 504, 505, 506, 510, 535, 536, 537, 538, 540, 541, 542,550, 573, 574, 580 581, 582 and 584 in SEQ ID NO: 2 the variant albuminor fragments thereof, or fusion polypeptides comprising variant albuminor fragments thereof according to the invention may contain additionalsubstitutions, deletions or insertions in other positions of themolecules. Such additional substitutions, deletions or insertions may beuseful in order to alter other properties of the molecules such as butnot limited to altered glycosylation; introduction of reactive groups ofthe surface such a thiol groups, removing/generating a carbamoylationsite; etc.

Residues that might be altered in order to provide reactive residues onthe surface and which advantageously could be applied to the presentinvention has been disclosed in the unpublished patent application WO2010/092135 (Included by reference). Particular preferred residuesinclude the positions corresponding to positions in SEQ ID NO: 2.

As examples of alterations that can be made in SEQ ID NO: 2 or incorresponding positions in other albumins in order to provide a reactivethiol group on the surface includes alterations corresponding tofollowing alterations in SEQ ID NO: 2: L585C, D1C, A2C, D562C, A364C,A504C, E5050, T79C, E86C, D129C, D549C, A581C, D121C, E82C, S270C,A578C, L595LC, D1DC, A2AC, D562DC, A364AC, A504AC, E505EC, T79TC, E86EC,D129DC, D549DC, A581AC, A581AC, D121DC, E82EC, S270SC, A579AC, C360*,C316*, C75*, C168*, C558*, C361*, C91*, C124*, C169* and C567*.Alternatively a cysteine residue may be added to the N or C terminal ofalbumin.

Polynucleotides

The present invention also relates to isolated polynucleotides thatencode any of the variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more (several) control sequences that direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of a variant. Manipulation of the polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, which is recognized bya host cell for expression of the polynucleotide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the variant. The promoter may be any nucleic acid sequence that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaeprotease A (PRA1), Saccharomyces cerevisiae protease B (PRB1),Saccharomyces cerevisiae translation elongation factor (TEF1),Saccharomyces cerevisiae translation elongation factor (TEF2),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI),Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomycescerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeasthost cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the variant. Any terminator that is functionalin the host cell may be used.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), Saccharomyces cerevisiae alcohol dehydrogenase(ADH1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphatedehydrogenase. Other useful terminators for yeast host cells aredescribed by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′-terminus ofthe polynucleotide encoding the variant. Any leader sequence that isfunctional in the host cell may be used.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding region naturally linked in translation reading frame withthe segment of the coding region that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding region that is foreign to the coding sequence. Theforeign signal peptide coding region may be required where the codingsequence does not naturally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the variant. However, any signal peptide coding region thatdirects the expressed variant into the secretory pathway of a host cellmay be used.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

Where both signal peptide and propeptide regions are present at theN-terminus of a variant, the propeptide region is positioned next to theN-terminus of the variant and the signal peptide region is positionednext to the N-terminus of the propeptide region.

Methods of Production

The variants of the present invention can be prepared using techniqueswell known to the skilled person. One convenient way is by cloningnucleic acid encoding the parent albumin or a fragment thereof or fusionpolypeptide comprising albumin or a fragment thereof, modifying saidnucleic acid to introduce the desired substitution(s) at one or more(several) positions corresponding to positions 417, 464, 490, 492, 493,494, 495, 496, 499, 500, 501, 503, 504, 505, 506, 510, 535, 536, 537,538, 540, 541, 542, 550, 573, 574 and 580 in SEQ ID NO: 2, where thevariant is not the variant consisting of SEQ ID NO:2 with thesubstitution D494N, E501K, K541E, D550G,A, K573E or K574N, preparing asuitable genetic construct where the modified nucleic acid is placed inoperative connection with suitable regulatory genetic elements, such aspromoter, terminator, activation sites, ribosome binding sites etc.,introducing the genetic construct into a suitable host organism,culturing the transformed host organism under conditions leading toexpression of the variant and recovering the variant. All thesetechniques are known in the art and it is within the skills of theaverage practitioner to design a suitable method for preparing aparticular variant according to the invention.

The variant polypeptide of the invention may also be connected to asignal sequence in order to have the variant polypeptide secreted intothe growth medium during culturing of the transformed host organism. Itis generally advantageous to have the variant polypeptide secreted intothe growth medium in order to ease recovery and purification.

Techniques for preparing variant polypeptides have also been disclosedin WO 2009019314 (included by reference) and these techniques may alsobe applied to the present invention.

Albumins have been successfully expressed as recombinant proteins in arange of hosts including fungi (including but not limited to Aspergillus(WO06066595), Kluyveromyces (Fleer 1991, Bio/technology 9, 968-975),Pichia (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) andSaccharomyces (Sleep 1990, Bio/technology 8, 42-46)), bacteria(Pandjaitab 2000, J. Allergy Clin. Immunol. 105, 279-285)), animals(Barash 1993, Transgenic Research 2, 266-276) and plants (including butnot limited to potato and tobacco (Sijmons 1990, Bio/technology 8, 217and Farran 2002, Transgenic Research 11, 337-346). The variantpolypeptide of the invention is preferably produced recombinantly in asuitable host cell. In principle any host cell capable of producing apolypeptide in suitable amounts may be used and it is within the skillsof the average practitioner to select a suitable host cell according tothe invention. A preferred host organism is yeast, preferably selectedamong Saccharomycacae, more preferred Saccharomyces cerevisiae.

The variant polypeptides of the invention may be recovered and purifiedfrom the growth medium using a combination of known separationtechniques such as filtration, centrifugation, chromatography, andaffinity separation techniques etc. It is within the skills of theaverage practitioner to purify the variants of the invention using aparticular combination of such known separation steps. As an example ofpurification techniques that may be applied to the variants of thepresent invention can be mentioned the teaching of WO0044772.

The variant polypeptides of the invention may be used for delivering atherapeutically beneficial compound to an animal or a human individualin need thereof. Such therapeutically beneficial compounds include, butare not limited, to labels and readily detectable compounds for use indiagnostics, such as various imaging techniques; pharmaceutical activecompounds such as drugs, or specifically binding moieties such asantibodies. The variants of the invention may even be connected to twoor more different therapeutically beneficial compounds, e.g., anantibody and a drug, which gives the combined molecule the ability tobind specifically to a desired target and thereby provide a highconcentration of the connected drug at that particular target.

Fusion Polypeptides

The variants of albumin or fragments thereof according to the inventionmay also be fused with a non-albumin polypeptide fusion partner. Thefusion partner may in principle be any polypeptide but generally it ispreferred that the fusion partner is a polypeptide having therapeutic ordiagnostic properties. Fusion polypeptides comprising albumin orfragments thereof are known in the art. It has been found that suchfusion polypeptide comprising albumin or a fragment thereof and a fusionpartner polypeptide have a longer plasma half-life compared to theunfused fusion partner polypeptide. According to the invention it ispossible to alter the plasma half-life of the fusion polypeptidesaccording to the invention compared to the corresponding fusionpolypeptides of the prior art.

One or more therapeutic polypeptides may be fused to the N-terminus, theC-terminus of albumin, inserted into a loop in the albumin structure orany combination thereof. It may or it may not comprise linker sequencesseparating the various components of the fusion polypeptide.

Teachings relating to fusions of albumin or a fragment thereof are knownin the art and the skilled person will appreciate that such teachingscan also be applied to the present invention. WO 2001/79271 A and WO2003/59934 A also contain examples of therapeutic polypeptides that maybe fused to albumin or fragments thereof, and these examples apply alsoto the present invention.

Conjugates

The variants of albumin or fragments thereof according to the inventionmay be conjugated to a second molecule using techniques known within theart. Said second molecule may comprise a diagnostic moiety, and in thisembodiment the conjugate may be useful as a diagnostic tool such as inimaging; or the second molecule may be a therapeutic compound and inthis embodiment the conjugate may be used for therapeutic purposes wherethe conjugate will have the therapeutic properties of the therapeuticcompound as well as the long plasma half-life of the albumin. Conjugatesof albumin and a therapeutic molecule are known in the art and it hasbeen verified that such conjugates have long plasma half-life comparedwith the non-conjugated, free therapeutic molecule as such. Theconjugates may conveniently be linked via a free thio group present onthe surface of HSA (amino acid residue 34 of mature HSA) using wellknown chemistry.

In one particular preferred aspect the variant albumin or fragmentthereof is conjugated to a beneficial therapeutic compound and theconjugate is used for treatment of a condition in a patient in needthereof, which condition is responsive to the particular selectedtherapeutic compound. Techniques for conjugating such a therapeuticallycompound to the variant albumin or fragment thereof are known in theart. WO 2009/019314 discloses examples of techniques suitable forconjugating a therapeutically compound to a polypeptide which techniquescan also be applied to the present invention. Further WO 2009/019314discloses examples of compounds and moieties that may be conjugated tosubstituted transferrin and these examples may also be applied to thepresent invention. The teaching of WO 2009/019314 is included herein byreference.

HSA contains in its natural form one free thiol group that convenientlymay be used for conjugation. As a particular embodiment within thisaspect the variant albumin or fragment thereof may comprise furthermodifications provided to generate additional free thiol groups on thesurface. This has the benefit that the payload of the variant albumin orfragment thereof is increased so that more than one molecule of thetherapeutic compound can be conjugated to each molecule of variantalbumin or fragment thereof, or two or more different therapeuticcompounds may be conjugated to each molecule of variant albumin orfragment thereof, e.g., a compound having targeting properties such asan antibody specific for example a tumour; and a cytotoxic drugconjugated to the variant albumin or fragment thereof thereby creating ahighly specific drug against a tumour. Teaching of particular residuesthat may be modified to provide for further free thiol groups on thesurface can be found in copending patent application WO 2010/092135,which is incorporated by reference.

Associates

The variants of albumin or fragments thereof may further be used in formof “associates”. In this connection the term “associate” is intended tomean a compound comprising a variant of albumin or a fragment thereofand another compound bound or associated to the variant albumin orfragment thereof by non-covalent binding. As an example of such anassociate can be mentioned an associate consisting variant albumin and alipid associated to albumin by a hydrophobic interaction. Suchassociates are known in the art and they may be prepared using wellknown techniques. As an example of a preferred associate according tothe invention can be mentioned an associate comprising variant albuminand paclitaxel.

Other Uses

The variant albumin or fragments thereof or fusion polypeptidescomprising variant albumin or fragments thereof according to theinvention have the benefit that their plasma half-life is alteredcompared to the parent albumin or fragments thereof or fusionpolypeptides comprising parent albumin or fragments thereof. This hasthe advantage that the plasma half-life of conjugates comprising variantalbumin or a fragment thereof or fusion polypeptide comprising variantalbumin or a fragment thereof, or an associate comprising variantalbumin or a fragment thereof according to the invention can be selectedin accordance with the particular therapeutic purpose.

For example for a conjugate, associate or fusion polypeptide used forimaging purposes in animals or human beings, where the imaging moietyhas an very short half-life and a conjugate or a fusion polypeptidecomprising HSA has a plasma half-life that is far longer than needed forthe imaging purposes it would be advantageous to use a variant albuminor fragment thereof of the invention having a shorter plasma half-lifethan the parent albumin or fragment thereof, to provide conjugates offusion polypeptides having a plasma half-life that is sufficiently longfor the imaging purpose but sufficiently short to be cleared form thebody of the particular patient on which it is applied.

In another example for a conjugate, an associate or fusion polypeptidecomprising a therapeutic compound effective to treat or alleviate aparticular condition in a patient in need for such a treatment it wouldbe advantageous to use the variant albumin or fragment thereof having alonger plasma half-life than the parent albumin or fragment thereof, toprovide associates or conjugates or fusion polypeptides having longerplasma half-lives which would have the benefit that the administrationof the associate or conjugate or fusion polypeptide of the inventionwould be needed less frequently or reduced dose with less side affectscompared to the situation where the parent albumin or associates thereofor fragment thereof was used.

In a further aspect the invention relates to compositions comprising thevariant albumin, associates thereof or fragment thereof, variant albuminfragment or associates thereof or fusion polypeptide comprising variantalbumin or fragment thereof according to the invention. The compositionsare preferably pharmaceutical compositions. The composition may beprepared using techniques known in the area such as disclosed inrecognized handbooks within the pharmaceutical field.

In a particular embodiment the compositions comprise a variant albuminor a fragment thereof according to the invention and a compoundcomprising a pharmaceutically beneficial moiety and an albumin bindingdomain (ABD). According to the invention ABD means a site, moiety ordomain capable of binding to circulating albumin in vivo and therebyconferring transport in the circulation of the ABD and any compound ormoiety bound to said ABD. ABD's are known in the art and have been shownto bind very tight to albumin so a compound comprising an ABD bound toalbumin will to a certain extent behave as a single molecule. Theinventors have realized by using the variant albumin or fragment thereofaccording to the invention together with a compound comprising apharmaceutically beneficial moiety and an ABD makes it possible to alterthe plasma half-life of the compound comprising a pharmaceuticallybeneficial moiety and an ABD compared to the situation where saidcompound were injected as such in a patient having need thereof oradministered in a formulation comprising natural albumin or a fragmentthereof.

The variant albumin or fragments thereof, conjugates comprising variantalbumin or a fragment thereof or fusion polypeptide comprising variantalbumin or a fragment thereof, or an associate comprising variantalbumin or a fragment thereof according to the invention may also beincorporated into nano- or microparticles using techniques well knownwithin the art. A preferred method for preparing nano- or microparticlesthat may be applied to the variant albumins or fragments thereofaccording to the invention is disclosed in WO 2004/071536, which isincorporated herein by reference.

Compositions

The present invention is also directed to the use of a variant ofalbumin or a fragment thereof or fusion polypeptides comprising variantalbumin or fragments thereof, or a conjugate comprising a variant ofalbumin or a fragment thereof, or an associate comprising a variant ofalbumin or a fragment thereof for the manufacture of a pharmaceuticalcomposition, where in the variant of albumin or a fragment thereof orfusion polypeptides comprising variant albumin or fragments thereof, ora conjugate comprising a variant of albumin or a fragment thereof, or anassociate comprising a variant of albumin or a fragment thereof has analtered plasma half-life compared with HSA or the corresponding fragmentthereof or fusion polypeptide comprising HSA or fragment thereof orconjugate comprising HSA.

In this connection the corresponding fragment of HSA is intended to meana fragment of HSA that aligns with and has same number of amino acids asthe fragment of the variant albumin with which it is compared. Similarlythe corresponding fusion polypeptide comprising HSA or conjugatecomprising HSA is intended to mean molecules having same size and aminoacid sequence as the fusion polypeptide of conjugate comprising variantalbumin, with which it is compared.

Preferably the variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, fragmentthereof, or a conjugate comprising a variant of albumin or a fragmentthereof has a plasma half-life that is higher than the plasma half-lifeof HSA or the corresponding fragment thereof or fusion polypeptidecomprising HSA or fragment thereof.

Alternatively, this may be expressed as the variant of albumin or afragment thereof or fusion polypeptides comprising variant albumin orfragments thereof, fragment thereof, or a conjugate comprising a variantof albumin or a fragment thereof has a KD to FcRn that is lower that thecorresponding KD for HSA or the corresponding fragment thereof or fusionpolypeptide comprising HSA or fragment thereof. Preferably, is KD forthe variant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof, or aconjugate comprising a variant of albumin or a fragment thereof lessthan 0.9×KD for HSA, more preferred less than 0.5×KD for HSA, morepreferred less than 0.1×KD for HSA, even more preferred less than0.05×KD for HSA, even more preferred less than 0.02×KD for HSA and mostpreferred less than 0.01×KD for HSA.

The variant of albumin or a fragment thereof or fusion polypeptidescomprising variant albumin or fragments thereof, fragment thereof, or aconjugate comprising a variant of albumin or a fragment thereof ispreferably the variant of albumin or a fragment thereof or fusionpolypeptides comprising variant albumin or fragments thereof, fragmentthereof, or a conjugate comprising a variant of albumin or a fragmentthereof according to the invention.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods ELISA:

Wells were coated with wild-type HSA or variants diluted in phosphatebuffered saline (PBS) to stated concentrations, incubated overnight at 4C and then blocked with 4% skimmed milk (Acumedia) for 1 hour at roomtemperature. The wells were then washed four times with PBS/0.005%TWEEN® 20 (PBS/T) pH 6.0 before glutathione-S-transferase (GST)-fused ()hFcRn (0.5 μg/ml) as described in FEBS J. 2008 August;275(16):4097-110. pre-incubated with an horseradish peroxidase(HRP)-conjugated polyclonal anti-GST from goat (1:5000; GE Healthcare),diluted in 4% skimmed milk PBS/0.005% TWEEN® 20 (PBS/T) pH 6.0 was addedto each well and incubated for 1.5 h at room temperature followed bywashing four times with PBS/T pH 6.0. One hundred μl of the substratetetramethylbenzidine (TMB) (Calbiochem) was added to each well andincubated for 45 min before 100 μl of 0.25 M HCl was added. Theabsorbance was measured at 450 nm using a Sunrise TECANspectrophotometer (TECAN, Maennedorf, Switzerland). The same ELISA wasrepeated with PBS/T pH 7.4.

Surface Plasmon Resonance (SPR):

SPR experiments were carried out using a Biacore 3000 instrument (GEHealthcare). Flow cells of CM5 sensor chips were coupled with shFcRn-GST(˜1400-5000 RU) using amine coupling chemistry as described in theprotocol provided by the manufacturer. The coupling was performed byinjecting 10 μg/ml of the protein in 10 mM sodium acetate pH 5.0 (GEhealthcare). Phosphate buffer (67 mM phosphate buffer, 0.15M NaCl,0.005% TWEEN® 20) at pH 6.0) was used as running buffer and dilutionbuffer. Regeneration of the surfaces were achieved using injections ofHBS-EP buffer (0.01M HEPES, 0.15M NaCl, 3 mM EDTA, 0.005% surfactantP20) at pH 7.4 (Biacore AB). For binding to immobilized shFcRn-GST,1.0-0.5 μM of each HSA variant was injected over the surface at constantflow rate (40 μl/ml) at 25 C. In all experiments, data was zero adjustedand the reference cell subtracted. Data evaluation was performed usingBIAevaluation 4.1 software (BIAcore AB).

The same SPR assay was repeated with HBS-EP buffer pH 7.4.

For the Purposes of this Patent Unless Otherwise Stated HSA, WT HSA, rHARefer to

Recombinant human serum albumin commercially available under theregistered tradename RECOMBUMIN (available from Novozymes Biopharma UKLtd, Nottingham UK) was used for the examples.

Serum Albumin from Other Species:

The albumins were recombinant wheres stated, produced using sequencesprovided from publicly available databases. Or purchased from commercialsuppliers.

FcRn

Expression and purification of soluble Human (shFcRn) and Mouse (smFcRn)FcRn: Methods for the generation of shFcRn and smFcRn expressionplasmids, expression and purification of each heterodimer can be foundin Berntzen et al. (2005) J. Immunol. Methods 298:93-104). AlternativelyshFcRn FcRn heterodimer was produced by GeneArt AG (Germany). Sequencesfor the two sub units of the heterodimer can be found in SEQ ID NO: 3and SEQ ID NO: 4. The soluble receptor was expressed in HEK293 cells andpurified from culture supernatant using Ni-HiTrap chromatographycolumns.

Example 1. Preparation of Variants Preparation of Specific HSA MuteinExpression Plasmids

Methods for the expression of HSA mutant variants and HSA fusionvariants were produced using several techniques. Standard molecularbiology techniques were employed throughout such as described inSambrook, J. and D. W. Russell, 2001. Molecular Cloning: a laboratorymanual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.

Method 1. Amino Acid Substitutions in HSA Detailed in Table1

Synthetic DNA NcoI/SacI fragments (859 bp) were generated by geneassembly (GeneArt AG, Germany) containing point mutations within theHSA-encoding gene (SEQ ID NO: 1) to introduce the desired amino acidsubstitution in the translated protein. Table 2 details the codons usedto introduce the amino acid substitutions into the HSA-encoding gene.The nucleotide sequence of the synthetic fragment encoding unchangedamino acids (i.e. wild type) was identical to that in pDB2243 (describedin WO 00/44772). The synthetic nucleotide fragments were ligated intoNcoI/SacI-digested pDB2243 to produce plasmids pDB3876-pDB3886 (Table1). For the production of expression plasmids, pDB3876-pDB3886 (seeTable 1) were each digested with NotI and PvuI, the DNA fragments wereseparated through a 0.7% (w/v) TAE gel, and 2992 bp fragments (NotIcassettes' including PRB1 promoter, DNA encoding the fusion leader (FL)sequence (disclosed in WO 2010/092135), nucleotide sequence encoding HSAand ADH1 terminator; see FIG. 1) were purified from the agarose gelusing a Qiagen Gel Extraction Kit following the manufacturer'sinstructions. ‘NotI cassettes’ were ligated into a NotI/Shrimp AlkalinePhosphatase (Roche)-treated “disintegration” plasmid pSAC35, disclosedin EP-A-286 424 and described by Sleep, D., et al. (1991) Bio/Technology9, 183-187. Ligation mixtures were used to transformchemically-competent E. coli DH5a. Expression plasmids pDB3887-pDB3897,pSAC35-derivatives containing the “NotI cassettes”, were identifiedusing standard techniques. Disintegration plasmids pDB3887-pDB3897 andpDB2244 (For the expression of wild type HSA, described in WO 00/44772)(Table 1) were used to transform S. cerevisiae BXP10cir⁰ (as previouslydescribed WO/2001/079480 as described below.

TABLE 1 Plasmid, amino acid substitution introduced into HSA PlasmidConstruct pDB2244 HSA pDB3876 HSA D494N pDB3877 HSA D494A pDB3878 HSAE495Q pDB3879 HSA E495A pDB3880 HSA D494Q pDB3881 HSA D494N, T496ApDB3882 HSA T496A pDB3883 HSA E492G pDB3884 HSA E492G, V493P pDB3885 HSAE492P pDB3886 HSA E492H pDB3887 HSA D494N pDB3888 HSA D494A pDB3889 HSAE495Q pDB3890 HSA E495A pDB3891 HSA D494Q pDB3892 HSA D494N, T496ApDB3893 HSA T496A pDB3894 HSA E492G pDB3895 HSA E492G, V493P pDB3896 HSAE492P pDB3897 HSA E492H n/a = Not applicable. pDB3876-pDB3886 aresub-cloning plasmids.

TABLE 2 Codons used to introduce amino acid substitutions into HSA Aminoacid Codon Gly GGT Glu GAA Asp GAT Val GTT Ala GCT Arg AGA Lys AAA AsnAAT Met ATG Ile ATT Thr ACT Trp TGG Cys TGT Tyr TAT Leu TTG Phe TTT SerTCT Gln CAA His CAT Pro CCA Stop TAA

Method 2. Production of HSA Variants D494N+E495Q+T496A and E495Q+T496A

A PCR-based method, using a QuickChange Lightening Kit (Statagene), wasemployed to introduce point mutations into HSA. Oligonucleotide pairsxAP094 (SEQ ID NO: 5)/xAP095 (SEQ ID NO: 6) and xAP096 (SEQ ID NO:7)/xAP097 (SEQ ID NO: 8) were used to generate two HSA variants(D494N+E495Q+T496A and E495Q+T496A, respectively). Plasmid pDB3927(disclosed in WO 2010/092135) was used as template DNA and themethodology recommended by the manufacturer of the kit was followed. Theresulting plasmids were named pDB3995 and pDB3996 (contain HSAD494N+E495Q+T496A and E495Q+T496A expression cassettes, respectively).pDB3995 and pDB3996 were digested with BstEII/BsrBI and the linearisedDNA molecules were purified using standard techniques. One hundred ng ofeach BstEII/BsrBI digested DNA, purified using a Qiagen PCR-Purificationkit following the manufacturer's instructions, was mixed individuallywith 100 ng Acc65I/BamHI-digested pDB3936) (disclosed in WO 2010/092135)and used to directly transform S. cerevisiae BXP10cir⁰ using the SigmaYeast Transformation kit described below.

Method 3. Amino Acid Substitutions in HSA Detailed in Table 3

Plasmid pDB3927 (disclosed in WO 2010/092135) (containing an identicalnucleotide sequence encoding HSA as in pDB2243) was manipulated to aminoacid substitutions within the mature HSA protein. Synthetic DNAfragments were generated (GeneArt AG, Germany or DNA2.0 Inc, USA)(NcoI/Bsu36I, AvrII/SphI or SacI/SphI fragments), containing pointmutations within the HSA-encoding gene to introduce the desired aminoacid substitution(s) into the translated protein sequence. Table 2details the codons used to introduce the amino acid substitutions intothe HSA-encoding gene. The nucleotide sequence of the synthetic fragmentencoding unchanged amino acids (i.e. wild type) was identical to thosein pDB3927. Synthetic DNA fragments were sub-cloned into NcoI/Bsu36I,AvrII/SphI-, SacI/Sph-digested pDB3927 (described in PCT 11527.204-WO)to generate pDB4006-pDB4010, pDB4083-pDB4101 and pDB4103-pDB4111 andpDB4194, pDB4200, pDB4202 (see Table 3).

Similarly, BamHI/SalI fragments containing point mutations in thenucleotide sequence encoding HSA were generated by gene assembly (DNA2.0Inc, USA) and ligated into BamHI/SalI-digested pDB3964 (described in WO2010/092135) to produce plasmids pDB3986-pDB3989 (Table 3).

The C-terminal string of amino acids from position 573-585(KKLVAASQAALGL) (SEQ ID NO: 9) in HSA were mutated to those in macaque(PKFVAASQAALA) (SEQ ID NO: 10), mouse (PNLVTRCKDALA) (SEQ ID NO: 11),rabbit (PKLVESSKATLG) (SEQ ID NO: 12) and sheep (PKLVASTQAALA) (SEQ IDNO: 13) serum albumin. The codons used to introduce each amino acidsubstitution are given in Table 2. Synthetic DNA fragments (SacI/SphI)were generated (DNA2.0 Inc, USA) by gene assembly (the nucleotidesequence of the synthetic fragment encoding unchanged amino acids (i.e.wild type) was identical to that in pDB3927) and were sub-cloned intoSacI/SphI-digested pDB3927 to produce plasmids pDB4114-4117 (Table 3).

Plasmids pDB3883 (Table 1), pDB4094 and pDB4095 (Table 3) were digestedwith NcoI/SacI and 857 bp fragments from each digest were purifiedbefore being ligated into NcoI/SacI-digested pDB4006 or pDB4110 (8.688kb) (Table 3) to produce pDB4156-pDB4161.

Expression plasmids were generated in vivo (i.e. via homologousrecombination in S. cerevisiae; a technique referred to as gap repair orin vivo cloning—see Orr-Weaver & Szostak. 1983. Proc. Natl. Acad. Sci.USA. 80:4417-4421). Modified plasmids listed in Table 3 were digestedwith BstEII/BsrBI and the linearised DNA molecules were purified usingstandard techniques. One hundred ng of each BstEII/BsrBI digested DNA,purified using a Qiagen PCR-Purification kit following themanufacturer's instructions, was mixed individually with 100 ngAcc65I/BamHI-digested pDB3936 (disclosed in WO 2010/092135) and used todirectly transform S. cerevisiae BXP10cir⁰ using the Sigma YeastTransformation kit described below.

TABLE 3 Plasmid Amino acid substitution in HSA pDB3986 HSA H440Q pDB3987HSA H464Q pDB3988 HSA H510Q pDB3989 HSA H535Q pDB4006 HSA K573A pDB4007HSA E492T/N503K/K541A pDB4008 HSA K541G pDB4009 HSA K541D pDB4010 HSAD550N pDB4083 HSA D494E/Q417H pDB4084 HSA Q417A pDB4085 HSA P499ApDB4086 HSA K500A pDB4087 HSA K536A pDB4088 HSA P537A pDB4089 HSA K538ApDB4090 HSA E492G/V493P/K538H/K541N/E542D pDB4091 HSAE492P/N503K/K541G/E542P pDB4092 HSA N503K pDB4093 HSA N503H pDB4094 HSAE492G/N503K pDB4095 HSA E492G/N503H pDB4096 HSA E492T pDB4097 HSA N503DpDB4098 HSA E492T/N503D pDB4099 HSA K538H pDB4100 HSA K541A pDB4101 HSAK541N pDB4103 HSA E542D pDB4104 HSA E542P pDB4105 HSA D550E pDB4106 HSAE492H/E501P/N503H/E505D/T506S/T540S/K541E pDB4107 HSAA490D/E492T/V493L/E501P/N503D/A504E/E505K/ T506F/K541D pDB4108 HSA E501ApDB4109 HSA E501Q pDB4110 HSA K573P pDB4111 HSA E492G/K538H/K541N/E542DpDB4114 HSA K573P/L575F/G584A pDB4115 HSAK573P/K574N/A577T/A578R/S579C/Q580K/A581D/ G584A pDB4116 HSAK573P/A577E/A578S/Q580K/A582T pDB4117 HSA K573P/A578S/S579T/G584ApDB4156 HSA E492G K573A pDB4157 HSA E492G N503K K573A pDB4158 HSA E492GN503H K573A pDB4159 HSA E492G K573P pDB4160 HSA E492G N503K K573PpDB4161 HSA E492G N503H K573P pDB4194 HSA D550E pDB4200 HSA K574NpDB4202 HSA Q580K

TABLE 4 K500 primers and plasmids CODONS Original primers USED xAP216CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Gly GGT (SEQ ID NO: 14) xAP217CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Glu GAA (SEQ ID NO: 15) xAP218CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Asp GAC (SEQ ID NO: 16) xAP219CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Val GTT (SEQ ID NO: 17) xAP220CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Arg AGA (SEQ ID NO: 18) xAP221CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Asn AAC (SEQ ID NO: 19) xAP222CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Met ATG (SEQ ID NO: 20) xAP223CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Ile ATT (SEQ ID NO: 21) xAP224CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Thr ACC (SEQ ID NO: 22) xAP225CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Trp TGG (SEQ ID NO: 23) xAP226CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Cys TGT (SEQ ID NO: 24) xAP227CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Tyr TAC (SEQ ID NO: 25) xAP228CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Leu TTG (SEQ ID NO: 26) xAP229CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Phe TTC (SEQ ID NO: 27) xAP230CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Ser TCT (SEQ ID NO: 28) xAP231CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Gln CAA (SEQ ID NO: 29) xAP232CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG His CAC (SEQ ID NO: 30) xAP233CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG Pro CCA (SEQ ID NO: 31) xAP234CTTTGGAAGTCGACGAAACTTACGTTCCA

GAATTCAACGCTG STOP taa (SEQ ID NO: 32) xAP235 GAATT

ATTACAAACCCAAAGCAGCTTGGGAAGC (SEQ ID NO: 33)

TABLE 5 K573 primers and plasmids CODONS Original primers USED xAP187ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Gly GGT (SEQ ID NO: 34) xAP188ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Glu GAA (SEQ ID NO: 35) xAP189ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Asp GAT (SEQ ID NO: 36) xAP190ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Val GTT (SEQ ID NO: 37) xAP191ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Arg AGA (SEQ ID NO: 38) xAP192ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Asn AAT (SEQ ID NO: 39) xAP193ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG  Met ATG (SEQ ID NO: 40) xAP194ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Ile ATT (SEQ ID NO: 41) xAP195ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Thr ACT (SEQ ID NO: 42) xAP196ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Trp TGG (SEQ ID NO: 43) xAP197ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Cys TGT (SEQ ID NO: 44) xAP198ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Tyr TAT (SEQ ID NO: 45) xAP199ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Leu TTG (SEQ ID NO: 46) xAP200ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Phe TTT (SEQ ID NO: 47) xAP201ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Ser TCT (SEQ ID NO: 48) xAP202ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG Gln CAA (SEQ ID NO: 49) xAP203ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG His CAT (SEQ ID NO: 50) xAP204ATAAGCCTAAGGCAGCTTGACTTGCAGCAACAAGTTT

ACCCTCCTCG STOP taa (SEQ ID NO: 51) xAP205 AATGCTG

AGATCTGCTTGAATGTGCTGATG (SEQ ID NO: 52)

Method 4. HSA K500 and K573 Permutation Library

PCR was used to produce two permutation libraries in which the codonsencoding amino acid 500 or 573 of mature HSA were changed (mutated) toalternative non-wild type amino acids and a termination codons(K5XXSTOP). Mutagenic oligonucletides (Table 4 and Table 5), weredesigned to amplify HSA-encoding DNA and incorporate the desiredchanges. That is, for the changes at position 500, pDB4082 (FIG. 1) wasused as a template DNA. pDB4082 is a derivative of pDB2305 (disclosed inEP1788084) and was produced as follows. pDB2305 (FIG. 2) was digestedwith NsiI/SpeI and the yielded 8.779 kb NsiI fragment was self-ligatedto produce pDB4005 (FIG. 3). A synthetic DNA fragment (BsaI/SphI) wasgenerated by gene assembly (DNA2.0 Inc, USA) (SEQ ID NO: 1) (containing3′ region of the PRB1 promoter, modified fusion leader sequence,nucleotide sequence encoding HSA and 5′ region of the modified ADH1terminator), and ligated into HindIII/SphI-digested pDB4005 (FIG. 3) toproduce pDB4082. Note. The HindIII site in PRB1 promoter site has beenremoved and a SaclI site within the nucleotide sequence encoding HSA hasbeen introduced.

For the permutation library for position 500 of HSA, the nucleotidesequence encoding HSA corresponding to that between the SalI/HindIIIsites (see plasmid map pDB4082, FIG. 1) was generated using the NewEngland Biolabs Phusion kit (Table 6) and oligonucleotides listed inTable 4. Table 7 describes the PCR method employed.

The permutation library at amino acid position 573 in HSA was generatedusing pDB3927 as template DNA and involved amplifying thealbumin-encoding DNA corresponding to that between the NcoI and Bsu36Isites using oligonucleotides detailed in Table 5.

TABLE 7 PCR conditions: 98° C. for 2 min 1 cycle 98° C. for 10 sec 35cycles 57° C. for 30 sec 72° C. for 20 sec 72° C. for 5 min 1 cycle

For the albumin variants based at positions 500 and 573, eachPCR-product was purified using a Qiagen PCR-clean up kit (according tothe manufactures instructions), digested with SalI/HindIII (position 500library) or NcoI/Bsu36I (position 573 library). The digested DNAs werethen purified using a Qiagen PCR-clean up kit and ligated intoSalI/HindIII—or NcoI/Bsu36I—digested pDB4082 or pDB3927, respectively,replacing the equivalent native sequence. Ligations were transformedinto E. coli DH5α, subsequent plasmids isolated from transformants usinga Qiagen miniprep kit (according to the manufacturer's instructions) andthe correct constructs identified by restriction analysis. This produceda collection of plasmids, pDB4204-pDB4222 (position 500 library) pDB4173to pDB4190 (position 573 library), containing albumin genes whichdiffered only in their sequence corresponding to the codon for the aminoacid at position 500 or 573 Table 4 and 5, respectively). The specificchanges in each plasmid were confirmed by sequencing.

The resultants plasmids were used to generate expression plasmids andalbumin fusion producing yeast by in vivo cloning as described above.That is, S. cerevisiae was transformed using the Sigma YeastTransformation kit (described below), using a mixture of a 100 ngBstEII/BsrBI-digeste HSA variant containing plasmid and 100 ngAcc65I/BamHI digested pDB3936.

Transformation of S. cerevisiae

S. cerevisiae BXP10 cir⁰ (as previously described WO/2001/079480) orStrain A cir⁰ (described in WO/2005/061718) was streaked on to YEPDplates (1% (w/v) yeast extract, 2% (w/v) Bactopeptone, 2% (w/v)glucose), 1.5% agar) and allowed to grow for 4 days at 30° C. prior totransformation. One μg of whole plasmid (i.e. circular plasmids) or, forgap repair, 100 ng BstEII/BsrBI- or NsiI/PvuI-digested HSA variant orHSA variant fusion containing plasmid and 100 ng Acc65I/BamHI digestedpDB3936 were used to transform S. cerevisiae using a Sigma YeastTransformation kit using a modified lithium acetate method (Sigma yeasttransformation kit, YEAST-1, protocol 2; Ito et al. (1983) J.Bacteriol., 153, 16; Elble, (1992) Biotechniques, 13, 18). The protocolwas amended slightly by incubating the transformation at roomtemperature for 4 h prior to heat shock. Following heat shock, the cellswere briefly centrifuged before being resuspended in 200 μl 1M sorbitolthen spread over BMMD agar plates, the composition of BMMD is describedby Sleep et al., (2001), Yeast, 18, 403. Plates were incubated at 30° C.for 4 days before individual colonies were patched on to fresh BMMDplates. Yeast strain numbers are detailed in Table 1.

Stocks were prepared for each yeast strain as follows: BMMD broth wasinoculated with a heavy loop of each yeast patch and grown for 24 h at30° C. with orbital shaking at 200 rpm. Cells were harvested bycentrifugation at 1900×g for 5 min in a Sorval RT600 centrifuge, 15 mLsupernatant was removed and replaced by trehalose 40% (w/v). The cellswere resuspended and transferred to cyrovials (1 mL) for storage at −80°C.

Shake Flask Growth of S. cerevisiae

BMMD (recipe 0.17% (w/v) yeast nitrogen base without amino acid andammonium sulphate (Difco), 37.8 mM ammonium sulphate, 29 mM citric acid,142 mM disodium hydrogen orthophosphate dehydrate pH6.5, 2% (w/v)glucose) media (10 mL) was inoculated with each yeast strain and grownfor 12 h at 30° C. with orbital shaking at 200 rpm. An aliquot of eachstarter culture (4 mL) was used to inoculate 2×200 mL BMMD media andgrown for 36 h at 30° C. with orbital shaking at 200 rpm. Cells wereharvested by filtration through 0.2 μm vacuum filter membranes(Stericup, Millipore) including a GF-D prefilter (Whatman) and thesupernatant retained for purification.

Primary Concentration

Retained culture supernatant was concentrated using Tangential FlowFiltration using a Pall Filtron LV system fitted with a Omega 10KD(0.093 sq·m2) filter (LV Centramate™ cassette, Pall Filtron) with atransmembrane pressure of 20 psi and a recirculation rate of 180mL·min⁻¹.

Fermentation

Fed-batch fermentations were carried out in a 10 L Sartorius Biostat Cfermenter at 30° C.; pH was monitored and adjusted by the addition ofammonia or sulphuric acid as appropriate. The ammonia also provided thenitrogen source for the cultures. The level of dissolved oxygen wasmonitored and linked to the stirrer speed, to maintain the level at >20%of saturation. Inocula were grown in shake flasks in buffered minimalmedia (recipe). For the batch-phase the cultures was inoculated intofermenter media (approximately 50% of the fermenter volume) containing2% (w/v) sucrose. The feed stage was automatically triggered by a sharprise in the level of dissolved oxygen. Sucrose was kept atgrowth-limiting concentrations by controlling the rate of feed to a setnominal growth rate. The feed consisted of fermentation media containing50% (w/v) sucrose, all essentially as described by Collins. (Collins, S.H., (1990) Production of secreted proteins in yeast, in: T. J. R. Harris(Ed.) Protein production by biotechnology, Elsevier, London, pp. 61-77).

GP-HPLC Quantitation

Purified albumin variants, fusions and conjugates were analysed byGP-HPLC and quantification as follows. Injections of 25 μL were madeonto a 7.8 mm id×300 mm length TSK G3000SWXL column (Tosoh Bioscience),with a 6.0 mm id×40 mm length TSK SW guard column (Tosoh Bioscience).Samples were chromatographed in 25 mM sodium phosphate, 100 mM sodiumsulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL/min, Samples werequantified by UV detection at 280 nm, by peak area, relative to arecombinant human albumin standard of known concentration (10 mg/mL) andcorrected for their relative extinction coefficients.

Purification of Albumin Variants from Shake Flask

Albumin variants were purified from shake flask (either culturesupernatant or concentrated culture supernatant) using a singlechromatographic step using an albumin affinity matrix(AlbuPure™—ProMetic BioSciences, Inc.). Chromatography was performed ata constant linear velocity of 240 cm/h throughout. Culture supernatantwas applied to a 6 cm bed height, 2.0 mL packed bed pre-equilibratedwith 50 mM sodium acetate pH5.3. Following load the column was washedwith 10 column volume (CV) of equilibration buffer, then 50 mM ammoniumacetate pH8.0 (10CV). Product was eluted with either 50 mM ammoniumacetate 10 mM octanoate pH8.0, 50 mM Ammonium Acetate 30 mM SodiumOctanoate 200 mM Sodium Chloride pH7.0 or 200 mM Potassium thiocyanate.The column was cleaned with 0.5M NaOH (3 cv) and 20 mM NaOH (3.5 cv).Eluate fraction from each albumin variant were concentrated anddiafiltered against 10 volumes of 50 mM sodium chloride (Vivaspin2010,000 MWCO PES with optional diafiltration cups, Sartorius). Purifiedalbumin variants were quantified by GP-HPLC as described above.

Purification of Albumin-Fusion Variants from Shake Flask

Albumin-fusion variants were purified from shake flask culturesupernatant using a single chromatographic step using an albuminaffinity matrix (AlbuPure™—ProMetic BioSciences, Inc.). Chromatographywas performed at a constant linear velocity of 240 cm/h throughout.Culture supernatant or concentrated culture supernatant was applied to a6 cm bed height, 2.0 mL packed bed pre-equilibrated with 50 mM sodiumacetate pH5.3. Following load the column was washed with 10 columnvolume (cv) equilibration buffer then 50 mM ammonium acetate pH8.0 (10cv). Product was eluted with either 50 mM ammonium acetate 10 mMoctanoate pH8.0, 50 mM Ammonium Acetate 30 mM Sodium Octanoate 200 mMSodium Chloride pH7.0, 50 mM Ammonium Acetate 100 mM Sodium OctanoatepH9.0 or 200 mM Potassium thiocyanate. The column was cleaned with 0.5MNaOH (3 cv) and 20 mM NaOH (3.5 cv). Eluate fraction from each albuminvariant-fusion were concentrated and diafiltered against 10 volumes of25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4 (Vivaspin20 10,000 MWCO PESwith optional diafiltration cups, Sartorius). Purified albumin-fusionvariants were quantified by GP-HPLC as described above.

Purification of Albumin Variants from Fermentation

Albumin variants were purified from high cell density fed batchfermentation supernatants after separation by centrifugation, using aSorvall RC 3C centrifuge (DuPont). Culture supernatant waschromatographed through an 11 cm bed height column 8.6 mL packed bedpacked with a custom synthesised albumin affinity matrix(AlbuPure™—ProMetic BioSciences, Inc.) as described above. Product waseluted using elution buffers describe above at a flow rate of 120 cm/h.The eluate fraction(s) was analysed by GP-HPLC. (above). and reducingSDS-PAGE for purity and if required concentrated (Vivaspin20 10,000 MWCOPES) and applied to a 2.4×96 cm column packed with Superdex 75 run at aflow rate of 39 cm/h in 25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4. Thepeak was fractionated, assayed by GP-HPLC and pooled in order togenerate the monomeric protein of interest. Pooled fractions wereconcentrated (Vivaspin20 10,000 MWCO PES, Sartorius).

All proteins to be assayed for receptor (FcRn) binding properties and orother analysis were quantified by GP-HPLC as described above correctedfor their relative extinction coefficients.

Example 2. Determination of Receptor (shFcRn) Binding Properties ofBlood Derived HSA and Recombinant Human Albumin

Essentially fatty acid-free HSA (Sigma-Aldrich) was further purified bysize exclusion chromatography as described in Andersen et al (2010). J.Biol. Chem. 285, (7), 4826-4836. Ten μM of monomeric HSA and rHA wereanalysed using SPR as described above and the data presented in FIG. 4.

Direct comparison of HSA (blood derived) with recombinant human albumin(Recombumin) at the same concentration (10 μM) (FIGS. 4A and 4B) showsfor both samples binding to immobilized shFcRn (pH6.0, pH7.4respectively) was reversible and pH dependent. In addition, comparisonof HSA vs recombinant human albumin by Bosse et al (2005). J. Clin.Pharmacol. 45; 57-67, demonstrated equivalent half life in vivo humanstudy

Example 3. Determination of Receptor (shFcRn) Binding Properties ofAlbumin Variants

Two established FcRn binding assays were used, ELISA and SPR. There aremajor differences between the assays: In the ELISA system HSA is coateddirectly in wells and shFcRn-GST is added in solution whereas in the SPRassay shFcRn-GST is immobilized to a CM5 chip and HSA injected insolution. The pH can be varied in both systems.

The variants were analysed using ELISA at pH 6.0 and pH 7.4. Results aredisclosed in FIG. 5. The ELISA values represent the mean of duplicates.

The variants were analysed using SPR analysis at pH 6.0 and pH 7.4.Results are disclosed for a representative number of variants in FIG. 6using a concentration of the variants of 0.2 μM and in FIG. 7 using aconcentration of the variants of 1 μM.

The SPR data disclosed in FIGS. 6 and 7 were normalized and the relativebinding of variants at each concentration is shown in FIGS. 8 A and Brespectively.

The conclusions of the analysis are that all tested variants have thecharacteristic binding to the receptor at pH 6.0 but no binding at pH7.4. The variants D494N,Q,A, E495Q,A, T496A, and D494N+T496A showreduced binding to the receptor compared to HSA.

Example 4. Determination of Receptor (shFcRn/smFcRn) Binding Propertiesof Albumin Variants

Using the SPR analysis method below the association constant Ka, thedissociation constant Kd and the binding constant KD calculated for HSAand mouse serum albumin (MSA) binding to human and mouse FcRn (Table 8).

SPR Analyses—

SPR analyses were performed on a BIAcore 3000 instrument (GE Healthcare)using CM5 chips and immobilization of smFcRn-GST and shFcRn-GST variantsor smFcRn was performed using the amine coupling kit (GE Healthcare).Protein samples (10 μg/ml) were injected in 10 mM sodium acetate at pH4.5 (GE Healthcare), all as described by the manufacturer. Unreactedmoieties on the surface were blocked with 1 M ethanolamine. For allexperiments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl,0.005% TWEEN® 20) at pH 6.0 or pH 7.4, or HBS-P buffer (0.01 M HEPES,0.15 M NaCl, 0.005% surfactant P20) at pH 7.4 were used as runningbuffer or dilution buffer. Kinetic measurements were performed using alow density immobilized surface (100-200 resonance units (RU)). Serialdilutions of hIgG1 (2000.0-31.2 nM), mIgG1 (1000.0-15.6 nM), MSA(20.0-0.3 μM) and HSA (200.0-3.1 μM) were injected at pH 6.0 or pH 7.4,at a flow rate 50 μl/minute at 25° C. Additive binding was recorded byinjecting HSA (10 μM), MSA (5 μM), hIgG1 (100 nM) or mIgG1 (100 nM)alone or two at a time at 25° C. at 20 μl/minute at pH 6.0 overimmobilized shFcRn (˜600 RU) or smFcRn (˜600 RU). Competitive bindingwas measured by injecting shFcRn (50 nM) or smFcRn (100 nM) alone ortogether with different amounts of HSA or MSA (10.0-0.05 μM) overimmobilized HSA (˜2600 RU) or MSA (˜2000 RU). In all cases, to correctfor nonspecific binding and bulk buffer effects, responses obtained fromthe control surfaces and blank injections were subtracted from eachinteraction curve. Kinetic rate values were calculated using predefinedmodels (Langmuir 1:1 ligand model, heterogeneous ligand model and steadystate affinity model) provided by the BIAevaluation 4.1 software. Thecloseness of the fit, described by the statistical value χ² thatrepresents the mean square, was lower than 2.0 in all affinityestimations.

TABLE 8 Binding constants of HSA and MSA shFcRn and smFcRn. KD AlbuminFcRn Ka Kd KD Req. Species Species (10³/Ms) (10³/s) (μM) (μM) MSA Mouse4.2 ± 0.5 39.4 ± 3.1 9.3 ± 0.4  ND^(d) MSA Human 3.8 ± 0.0  3.1 ± 0.10.8 ± 0.2 ND HSA Mouse NA NA NA 86.2 ± 4.1 HSA Human 2.7 ± 1.3 12.2 ±5.9 4.5 ± 0.1  4.6 ± 0.5 The KD's were generated using the BIAevaluation4.1 software) A Langmuir 1:1 ligand model was used throughout. Thekinetic values represent the average of triplicates. ND means: Notdetermined. NA means: Not acquired

Example 5. Binding of Albumins from Other Species to Human FcRn

Commercially available animal albumin (either Sigma-Aldrich orCalbiochem) were further purified as described in Andersen et al (2010).J. Biol. Chem. 285, (7), 4826-4836. The binding of donkey serum albumin,bovine serum albumin, goat serum albumin, sheep serum albumin, rabbitserum albumin, dog serum albumin, hamster serum albumin, guinea pigalbumin, rat serum albumin and chicken serum albumin to shFcRn wasdetermined using the techniques described in Materials and Methods. TheELISA results are disclosed in FIG. 9 A-D and the relative bindingssummarized in FIG. 9 E.

The SPR results are shown in FIG. 10, where the binding at pH 6.0 and pH7.4 for each albumin species are shown. Table 10 shows an overview ofthe relative binding responses measured using ELISA and SPR:

TABLE 10 Cross-species albumin-FcRn binding shFcRn Albumin ELISA SPRspecie pH 6.0 pH 7.4 pH 6.0 pH 7.4 Human ++(+) − ++(+) − Donkey +++ − ++− Cow ++ − ++ − Sheep +/− − − − Goat +/− − − − Rabbit ++++ − +++ − DogND^(a) ND +++ − G. pig ++++ + ++++ + Hamster +++ − +++ − Rat +++ − +++ −Mouse +++ − +++ − Chicken − − − − Relative binding responses arecategorized from strongest (++++) to weakest (+) and no binding (−).^(a)Not determined (ND).

A hierarchy ranging from strongest to weakest binding is as follows;guineapig=/>rabbit>hamster/dog>rat/mouse>donkey>human>bovine>goat/sheep>chicken.This data shows that animal albumins have different affinities forshFcRn.

Example 6. Kinetics of the HSA Variant for shFcRn

The binding constants for variants according to the invention weredetermined according to the methods described in Materials and Methods.

TABLE 11 Binding constants of HSA variants for shFcRn Albumin Ka kd KDKD Req Variant (10³/Ms) (10⁻³/s) (μM) (μM) WT 3.2 ± 0.2 15.5 ± 2.5 4.85.4 D494N 1.7 ± 0.0 18.6 ± 0.0 10.9 11.8 D494A 2.3 ± 0.1 53.4 ± 0.3 23.217.0 D494Q 2.1 ± 0.0 58.2 ± 3.8 27.7 ND E495Q 2.5 ± 0.0 24.1 ± 0.2 9.610.9 E495A 2.1 ± 0.0 14.0 ± 0.0 7.0 8.6 D494N + T496A 2.5 ± 0.0 11.0 ±0.0 4.4 5.5 T496A 2.3 ± 0.0 11.7 ± 0.5 5.1 7.1 E492G 4.1 ± 0.0 11.0 ±0.0 2.7 ND The KD's were generated using the BIAevaluation 4.1 software)A Langmuir 1:1 ligand model was used throughout. The kinetic valuesrepresent the average of triplicates. ND means: Not determined.

The results correspond with the conclusions made in Example 3 based onSPR and ELISA data but in addition shows that E492G has increasedaffinity to its receptor,

Example 7. Competitive Analysis of the HSA Variants

Competitive analysis of the HSA variants prepared in example 1 and WTHSA was performed using the methods described in example 4. Results areshown in FIG. 15. The results show that the variant E492G, unlike E492HE492P and E492G+V493P, has stronger binding to shFcRn than HSA.

Example 8. Analysis of Q417 Substitutions

Using the method of Example 1 variants of HSA having the substitutionsQ417A and D494E+Q417H were constructed. The kinetic properties of thesevariants were tested using the methods in Materials and Methods and areshown in Table 12.

TABLE 12 Binding constants of HSA variants for shFcRn Albumin ka kdKD^(b) KD Req^(c) variant^(a) (10³/Ms) (10⁻³/s) (μM) (μM) WT 3.2 ± 0.215.5 ± 2.5 4.8 5.4 Q417A 3.2 ± 0.1 26.0 ± 0.0 8.1 ND D494E + Q417H 3.1 ±0.1 20.5 ± 0.5 6.6 ND ^(a)Dilutions of HSA variants were injected overimmobilized shFcRn (~1500 RU). ^(b)The kinetic rate constants wereobtained using a simple first-order (1:1) bimolecular interaction model.^(c)The steady state affinity constant was obtained using an equilibrium(Req) binding model supplied by the BIAevaluation 4.1 software. Thekinetic values represent the average of triplicates. d: Not determined(ND).

The data show that variants Q417A and D494E+Q417H bind weaker to thereceptor than the wild-type HSA.

Example 9. Analysis of HSA Variants in Position 499, 500, 536, 537, 538and 573

Using the method of Example 1 variants of HSA having the substitutionsP499A, K500A, K536A, P537A, K538A and K573A were constructed. Thereceptor binding properties of these variants were tested as describedin Materials and Methods. Results are shown in FIG. 11.

The data demonstrated that variants P499A, K536A, P537A and K538A had areduced binding affinity to shFcRn relative to HSA. Variant K500A hadalmost completely lost its ability to bind to shFcRn and K573A had anincreased binding affinity to shFcRn both relative to HSA.

Example 10. Analysis of Variants in Position 501 of HSA

Using the method of Example 1 variants of HSA having the substitutionsE501A and E501Q were constructed. The kinetic properties of thesevariants were tested as described in Materials and Methods.

TABLE 13 Binding constants of HSA variants for shFcR Albumin ka kdKD^(b) KD Req^(c) variant^(a) (10³/Ms) (10⁻³/s) (μM) (μM) WT 3.2 ± 0.215.5 ± 2.5 4.8 5.4 E501A 3.3 ± 0.0 26.0 ± 0.0 7.8 ND E501Q 2.7 ± 0.115.5 ± 0.5 5.7 ND ^(a)Dilutions of HSA variants were injected overimmobilized shFcRn (~1500 RU). ^(b)The kinetic rate constants wereobtained using a simple first-order (1:1) bimolecular interaction model.^(c)The steady state affinity constant was obtained using an equilibrium(Req) binding model supplied by the BIAevaluation 4.1 software. Thekinetic values represent the average of triplicates d: Not determined(ND).

The data shows that variants E501A and E501Q have a slightly decreasedbinding affinity to shFcRn relative to HSA.

Example 11. Analysis of HSA Variants in Position 573

Using the method of Example 1 variants of HSA having a substitution atposition 573 were constructed. All variants at position 573 weregenerated and the receptor binding properties of these variants weretested as described in Materials and Methods but with SPR analysisperformed at pH5.5. Results are shown in the table 14 below and FIGS. 12and 13.

TABLE 14 Kinetics of HSA K573 single point mutants. Albumin ka kd KD^(b)variant^(a) (10³/Ms) (10⁻³/s) (nM) WT 9.0 ± 0.0 6.9 ± 0.1 766 K573A 7.4± 0.0 2.2 ± 0.0 297 K573C 4.2 ± 0.0 1.1 ± 0.2 262 K573D 7.9 ± 0.2 4.1 ±0.3 518 K573E 9.0 ± 0.0 2.9 ± 0.0 322 K573F 7.8 ± 0.1 0.5 ± 0.1 74 K573G8.5 ± 0.0 1.8 ± 0.1 212 K573H 12.0 ± 0.2  0.8 ± 0.0 68 K573I 8.6 ± 0.00.8 ± 0.2 99 K573L 5.1 ± 0.2 2.3 ± 0.1 451 K573M 8.6 ± 0.0 1.9 ± 0.0 221K573N 7.3 ± 0.2 1.1 ± 0.3 151 K573P 9.8 ± 0.0 0.6 ± 0.1 61 K573Q 7.7 ±0.2 2.6 ± 0.0 338 K573R 8.5 ± 0.0 3.0 ± 0.2 353 K573S 7.9 ± 0.2 1.2 ±0.2 152 K573T 8.7 ± 0.2 1.1 ± 0.1 126 K573V 8.1 ± 0.0 0.6 ± 0.2 80 K573W15.0 ± 0.2  0.4 ± 0.3 29 K573Y 22.0 ± 0.1  0.5 ± 0.1 23 K573STOP ND ND141000 ^(a)Dilutions of HSA variants were injected over immobilizedshFcRn (~1500 RU). ^(b)The kinetic rate constants were obtained using asimple first-order (1:1) bimolecular interaction model. c: The steadystate affinity constant was obtained using an equilibrium (Req) bindingmodel supplied by the BIAevaluation 4.1 software. The kinetic valuesrepresent the average of duplicates. d: Not determined (ND).

The results show that all variants having substitution in position 573have improved binding to shFcRn compared with WT HSA. In particular thevariants K573F, K573H, K573P, K573W and K573Y have more than 10 foldlower KD to shFcRn than the parent HSA. The variant K573STOP is atruncated albumin having a stop codon in position 573. The sensorgramfor the K573STOP variant show significantly reduced binding compare tothe WT HSA and generated a high KD. The increased affinity that we haveshown for the variant K573E, a natural variant characterized by Otagiri(2009). Biol. Pharm. Bull. 32(4) 527-534, is predicted to have increasedhalf-life in vivo.

Example 12. Analysis of Further HSA Variants

Using the method of Example 1 variants of HSA having the substitutionsE492G, E492G+N503H, N503H, D550E, E492G+N503K, E542P, H440Q, K541G,K541D, D550N E492G+K538H+K541N+E542D, E492T+N503K+K541A,E492P+N503K+K541G+E542P, E492H+E501P+N503H+E505D+T506S+T540S+K541E,A490D+E492T+V493L+E501P+N503D+A504E+E505K+T506F+K541D,E492G+V493P+K538H+K541N+E542D were constructed. The receptor bindingproperties of these variants were tested as described in Materials andMethods, and the results are shown in Table 15 and FIG. 14.

TABLE 15 Binding constants of HSA variants for shFcR Albumin Ka kdKD^(b) KD Req^(c) variant^(a) (10³/Ms) (10⁻³/s) (μM) (μM) WT 3.2 ± 0.215.5 ± 2.5 4.8 5.4 E492G 4.1 ± 0.0 11.0 ± 0.0 2.7 ND E492G/N503H 6.9 ±0.1 14.5 ± 0.5 2.1 ND N503H 5.4 ± 0.0 24.0 ± 0.1 4.4 ND D550E 3.2 ± 0.411.8 ± 0.0 3.6 ND E492G/N503K 5.9 ± 0.1 16.0 ± 0.0 2.7 ND E542P 3.4 ±0.0 15.7 ± 0.2 4.7 ND H440Q 3.2 ± 0.1 20.8 ± 0.0 6.5 ND K541G 3.2 ± 0.023.0 ± 0.0 7.1 ND K541D 2.6 ± 0.0 24.0 ± 0.0 9.2 ND D550N 2.5 ± 0.0 30.0± 0.0 12.0 ND ^(a)Dilutions of HSA variants were injected overimmobilized shFcRn (~1500 RU). ^(b)The kinetic rate constants wereobtained using a simple first-order (1:1) bimolecular interaction model.^(c)The steady state affinity constant was obtained using an equilibrium(Req) binding model supplied by the BIAevaluation 4.1 software. Thekinetic values represent the average of triplicates. d: Not determined(ND).

The results show that for position 550, a substitution to E results inan increased affinity whilst a substitution to N resulted in reducedaffinity for shFcRn at pH6.0. When this analysis was repeated for theD550E substitution at pH5.5 however no observable increase in affinitywas seen. The substituted for an acid amino acid (E) maintains andimproves the binding. However the substitution for an uncharged amideamino acid reduces binding at pH6.0. Based on this observation, we wouldpredict for this position that substitutions to basic amino acids (H, Kand R) would result in further reductions in binding.

Example 13. Mutations in his Residues

The following variants were generated using the methods described inExample 1: H440Q, H464Q, H510Q and H535Q. FIG. 15 shows SPR sensorgramsof these variants interacting with shFcRn as described in Materials andMethods.

It was found that the variant H440Q bound with comparable affinity asHSA. In contrast H464Q, H510Q and H535Q had significantly reducedaffinity to shFcRn. This supports the previously published observationsthat mutagenesis of these Histidine residues significantly reduced HSAbinding to shFcRn (Wu et al (2010). PEDS, 23(10)789-798). Wu et al showa reduced half-life for a diabody fusion proteins (scFv-DIII)2 in micewith an order of removal from slowest to fastest: Db-DIIIWT>H535A>H510A>H464A>Db. Based on affinity to shFcRn and when comparedto smFcRn (example 5) we would predict the clearance order in humans tobe (for glutamine (Q) substitutions) WT>H440Q>H510Q>H464Q>H535Q.

Example 14. Further Variants

The following variants were generated using the methods described inExample 1: K574N and Q580K in HSA. Binding of the variants to FcRn wastested using the SPR assay as described in Materials and Methods and theresults are shown in Table 16.

The results show that variants K574N and Q580K bound stronger to shFcRn.

TABLE 16 Following kinetic data was found for these variants: Albumin kakd KD variant (10³/Ms) (10⁻³/s) (μM) WT 9.7 ± 0.0 30.0 ± 0.1  3.1 K574N4.9 ± 01  8.4 ± 0.1 1.7 Q580K 6.0 ± 0.0 9.3 ± 0.0 1.5

Example 15. Analysis of HSA Variants in Position 500

Using the method of Example 1 variants of HSA having a substitution atposition 500 were constructed. All variants at position 500 weregenerated and the receptor binding properties of these variants weretested. Biacore X, Biacore X100 and Sensor Chip CM5 were used for allanalyses, both supplied by G E Healthcare. shFcRn produced by GeneArt AG(Germany) (diluted to 10 μg/mL in 10 mM sodium acetate pH5.0 (G EHealthcare)) was immobilised on flow cell 2 (FC2) to levels between1600-2200 response units (RU) via standard amine coupling as permanufacturers instructions (G E Healthcare). A blank immobilisation wasperformed on flow cell 1 (FC1) for it to serve as a reference cell. Tostabilise the assay, 3-5 start up cycles were run first, with runningbuffer (67 mM phosphate buffer, 0.15M NaCl, 0.005% Tween 20 atpH5.75±0.25) only, followed by regeneration. WT rHA and K500 libraryvariants were injected at various concentrations (1 μM-150 μM) for 90 sat a constant flow rate of (30 μl/min) at 25° C. followed byregeneration of the surface using HBS-EP buffer pH7.4 (G E Healthcare)until approximate initial baseline RU was restored (usually 12 s pulsewould suffice).

Results are shown in the Table 17 and FIG. 16

TABLE 17 Kinetics of HSA K500 single point mutants. Albumin ka kd KD^(b)KD Req^(c) variant (10³/Ms) (10⁻³/s) (μM) (μM) K500R 4.42 7.21 1.63K500I 5.18 10.9 2.1 WT 4.24 9.2 2.2^(a) K500L 3.73 11.9 3.2 K500Q 1.073.4 3.2 K500V 3.29 11.0 3.3 K500Y 3.97 14.6 3.7 K500M 2.48 21.5 8.7K500T 1.2 13.4 11.2 K500W 0.5 5.4 11.7 K500N 1.3 18.2 14 K500F 5.17 73.714.3 K500H 4 63.8 16 K500P ND ND ND 51*   K500C 2.38 124 52 K500S ND ND - ND 70.2* K500A 2.61 208 79.9 K500D ND ND ND 83.3* K500G ND ND ND95.4* K500E KD not calculable see FIG. 16 K500 STOP Null binder ^(a)Meanof 4 values. ^(b)The kinetic rate constants were obtained using a simplefirst-order (1:1) bimolecular interaction model. ^(c)The steady stateaffinity constant was obtained using an equilibrium (Req) binding modelsupplied by the BIAevaluation 4.1 software.

The results show for variants K500R and K5001 have increased andcomparable affinity for shFcRn compared to WT HSA respectively. VariantK500E bound tightly to immobilised shFcRn but still demonstrated thecharacteristic pH-dependency of the FcRn interaction. This complex wasvery stable, such that kinetic analysis was not possible (FIG. 16). Allother variants have reduced binding to shFcRn than wt rHA.

All variants bound to shFcRn (to some extent) at pH5.5. No binding ofK500 library variants to shFcRn was detectable at pH7.4.

Example 16. Fusion Polypeptides

The generation of albumin fusions containing albumin muteins

Plasmids containing expression cassettes for the production of scFv(vHvL) genetically-fused to HSA, at either the N- or C-terminus or both,(described in, Evans et al., 2010. Protein Expression and Purification.73, 113-124) were modified to allow the production of albumin fusionsusing in vivo cloning (describe above). That is, pDB3017 (FIG. 17),pDB3021 (FIG. 18), pDB3056 (FIG. 19) were digested with NsiI/SpeI andNsiI fragments corresponding 9.511 kb, 9.569 kb and 8.795 kb,respectively, were purified using standard techniques. Purified NsiIfragments were self-ligated and used to transform chemically competentE. coli DH5α to produce pDB4168, pDB4169 and pDB4170, respectively(Table 18).

Similarly, pDB3165 (containing the bivalent fusion) (FIG. 20) wasdigested with NotI and the expression cassette (4.506 kb fragment) waspurified before being ligated into NotI-digested pDB3927 to producepDB4172 (FIG. 21, Table 18).

Synthetic SalI/Bsu36I DNA fragments (269 bp), which contain pointmutations within the albumin encoding nucleotide sequence to introduceamino acid substitutions corresponding to K500A, or D550N or K573P intothe translated albumin protein sequence, were generated by gene assembly(GeneArt AG, Germany). The SalI/Bsu36I fragments were individuallyligated into SalI/Bsu36I-digested pDB4168-pDB4170 and pDB4172 and usedto transform chemically competent E. coli DH5α using standard techniquesto generate plasmids pDB4265-pDB4276 (Table 18).

TABLE 18 Albumin variant fusions Plasmid Construct pDB3017 scFv(anti-FITC) - HSA - FLAG pDB3021 HSA - GS linker - scFv (anti-FITC) -FLAG pDB3056 HSA - FLAG pDB3165 scFv (anti-FITC) - HSA - GS linker -scFv (anti-FITC) - FLAG pDB4168 scFv (anti-FITC) - HSA - FLAG pDB4169HSA - GS linker - scFv (anti-FITC) - FLAG pDB4170 HSA - FLAG pDB4172scFv (anti-FITC) - HSA - GS linker - scFv (anti-FITC) - FLAG pDB4265scFv (anti-FITC) - HSA K500A - FLAG pDB4266 scFv (anti-FITC) - HSAD550N - FLAG pDB4267 scFv (anti-FITC) - HSA K573P - FLAG pDB4268 HSAK500A - GS linker - scFv (anti-FITC) - FLAG pDB4269 HSA D550N - GSlinker - scFv (anti-FITC) - FLAG pDB4270 HSA K573P - GS linker - scFv(anti-FITC) - FLAG pDB4271 HSA K500A - FLAG pDB4272 HSA D550N - FLAGpDB4273 HSA K573P - FLAG pDB4274 scFv (anti-FITC) - HSA K500A - GSlinker - scFv (anti- FITC) - FLAG pDB4275 scFv (anti-FITC) - HSA D550N -GS linker - scFv (anti- FITC) - FLAG pDB4276 scFv (anti-FITC) - HSAK573P - GS linker - scFv (anti- FITC) - FLAG pDB4277 scFv (anti-FITC) -HSA K573A - FLAG pDB4278 HSA K573A - GS linker - scFv (anti-FITC) - FLAGpDB4279 HSA K573A - FLAG pDB4280 scFv (anti-FITC) - HSA K573A - GSlinker - scFv (anti- FITC) - FLAG pDB4281 HSA K500A - GS linker - scFv(anti-FITC) pDB4282 HSA D550N - GS linker - scFv (anti-FITC) pDB4283 HSAK573P - GS linker - scFv (anti-FITC) pDB4284 HSA - GS linker - scFv(anti-FITC) pDB2613 HSA- GS linker -IL1RA (N84Q) pDB4285 HSA K573A- GSlinker -IL1RA (N84Q) pDB4286 HSA D550N- GS linker -IL1RA (N84Q) pDB4287HSA K500A- GS linker -IL1RA (N84Q) pDB4288 HSA K573P- GS linker -IL1RA(N84Q)

Similarly, a DNA fragment was generated by PCR (using standardtechniques), to introduce a K573A substitution in the translated albuminprotein sequence. PCR was performed using the New England BiolabsPhusion kit using pDB4267 (FIG. 22) as template DNA and oligonucleotidesxAP238 (SEQ ID NO: 53) and xAP239 (SEQ ID NO: 54):

Table 19 describes PCR cycling.

TABLE 19 PCR cycling 98° C. for 2 min 1 cycle 98° C. for 10 sec 35cycles 57° C. for 30 sec 72° C. for 10 sec 72° C. for 5 min 1 cycle

The PCR-product was purified, digested with SalI/Bsu36I, and thefragment (269 bp) isolated was ligated into SalI/Bsu36I-digestedpDB4168-pDB4170 and pDB4172 and used to transform chemically competentE. coli DH5α. Resulting plasmids (pDB4277-pDB4280) are listed in Table18.

The nucleotide sequence encoding the FLAG tag was removed from plasmidspDB4168 and pDB4268-4270 (plasmids for the expression of scFvN-terminally fused to HSA and HSA muteins K500A, D550N and K573P,respectively. pDB4168 and pDB4268-4270 (Table 18) were digested withBsu36I/SphI to remove a 231 bp product comprising 3′ region ofHSA-encoding gene, nucleotide sequence encoding FLAG tag and 5′ regionof ADH1 terminator. A Bsu36I/SphI fragment (207 bp), comprising 3′region of HSA-encoding gene and 5′ region of mADH1 terminator (SEQ ID1)from pDB4181 was ligated into Bsu36I/SphI-digested pDB4168 andpDB4268-pDB4270 using standard techniques. Ligation mixtures were usedto transform chemically competent E. coli DH5α using standard techniquesto generate plasmids pDB4281-pDB4284 (Table 18)

pDB4265-pDB4284 were digested with BstEII/BsrBI and the linearised DNAmolecules were purified using standard techniques. One hundred ngBstEII/BsrBI DNA samples were mixed with 100 ng Acc65I/BamHI-digestedpDB3936 and used to transform S. cerevisiae BXP10cir⁰ using the SigmaYeast Transformation kit described below. In each case the expressionplasmid was generated in the yeast by homologous recombination (in vivocloning) between the albumin-fusion containing plasmid (pDB4265-pDB4280)(Table 18) and pDB3936.

Plasmids pDB3017, pDB3021, pDB3056 and pDB3165 (wild type HSA fusions,described by Evans et al., 2010. Protein Expression and Purification.73, 113-124) were used to transform S. cerevisiae Strain Acir⁰(described in WO/2005/061718) using the Sigma Yeast Transformation kitdescribed below.

The nucleotide sequence encoding human IL-1RA (interleukin-1 receptorantagonist) (accession number: CAA59087) could be syntheticallygenerated by gene assembly. The nucleotide sequence of the 708 bpsynthetic fragment (Bsu36I/SphI fragment) is given in SEQ ID NO: 55 andincludes the 3′region of the gene encoding HSA, the nucleotide sequenceencoding a GS linker, the nucleotide sequence encoding human IL-1RA(N84Q to abolish the N-linked glycosylation motif) and the 5′ region ofthe ADH1 terminator. The synthetic DNA fragment could be ligated intoBsu36I/SphI-digested pDB3927 to produce pDB2588.

Plasmids containing the expression cassettes for the production ofIL-1RA genetically fused to the C-terminus of HSA and the HSA variantsK500A, D550N, K573A and K573P were prepared as follows. pDB2588 wasdigested with Bsu36I/SphI and a 705 bp fragment containing the ‘3 regionof the HSA encoding gene, nucleotide sequence encoding a GS linker,nucleotide sequence encoding human IL1-RA (N84Q) and the 5’ region of amodified S. cerevisiae ADH1 terminator (SEQ 1D3) was purified usingstandard techniques then ligated into Bsu36I/SphI-digested pDB4006(containing HSA K573A expression cassette), pDB4010 (containing HSAD550N expression cassette), pDB4086 (containing HSA K500A expressioncassette), pDB4110 (containing HSA K573P expression cassette) togenerate pDB4287, pDB4286, pDB4285 and pDB4288, respectively (for anexample, see FIG. 23). pDB4285-pDB4288 were digested with NsiI/PvuI andthe linearised DNA molecules were purified using standard techniques.One hundred ng NsiI/PvuI-digested DNA samples were mixed with 100 ngAcc65I/BamHI-digested pDB3936 (9721 bp) (i.e. in vivo cloning) and usedto transform S. cerevisiae (i.e. by in vivo cloning) using the SigmaYeast Transformation kit described below.

Preparation of an S. cerevisiae strain expressing wild type HSAgenetically fused to a GS linker and IL1-RA (N84Q) (see Table 18) couldalso be generated following the methods described above.

The fusion polypeptides were analysed for their binding to FcRn usingthe SPR method described above and following results were obtained:

TABLE 20 Kinetics of HSA fusion variants. Albumin ka kd KD^(b)variant^(a) (10³/Ms) (10⁻³/s) (μM) HSAWT 9.7 ± 0.0 30.0 ± 0.1  3.1 K574N4.9 ± 01  8.4 ± 0.1 1.7 Q580K 6.0 ± 0.0 9.3 ± 0.0 1.5 K573P 2.8 ± 0.00.4 ± 0.0 0.1 HSA-WT-FLAG 8.2 ± 0.2 24.0 ± 0.2  2.9 HSA-D550N-FLAG 5.9 ±0.0 49.0 ± 0.1  8.3 HSA-K500A-FLAG  ND^(c) ND ND HSA-K573A-FLAG 6.1 ±0.1 7.1 ± 0.1 1.1 HSA-K573P-FLAG 6.2 ± 0.1 1.2 ± 0.1 0.2 HSA-WT-IL1RA6.2 ± 0.0 25.0 ± 0.2  4.0 HSA-K500A-IL1RA ND ND ND HSA-D550N-IL1RA 7.3 ±0.2 38.0 ± 0.0  5.2 HSA-K573A-IL1RA 6.1 ± 0.0 7.1 ± 0.1 1.1HSA-K573P-IL1RA 6.2 ± 0.1 1.3 ± 0.1 0.2 scFv-HSA-K500A-FLAG ND ND NDscFv-HSA-D550N-FLAG 6.2 ± 0.0 18.0 ± 0.0  2.9 scFv-HSA-K573A-FLAG 6.4 ±0.1 5.7 ± 0.2 0.9 scFv-HSA-K573P-FLAG 5.8 ± 0.0 1.1 ± 0.1 0.2scFv-HSA-WT-scFv-FLAG 7.5 ± 0.0 15.0 ± 0.2  2.0 scFv-HSA-K500A-scFv-FLAGND ND ND scFv-HSA-D550N-scFv-FLAG 4.1 ± 0.1 27.0 ± 0.2  6.6scFv-HSA-K573P-scFv-FLAG 6.0 ± 0.2 0.7 ± 0.1 0.1 HSA-K500A-scFv-FLAG NDND ND HSA-D550N-scFv-FLAG 7.3 ± 0.1 42.0 ± 0.3  5.8 HSA-K573A-scFv-FLAG6.4 ± 0.1 5.7 ± 0.1 0.9 HSA-K573P-scFv-FLAG 4.7 ± 0.1 0.7 ± 0.1 0.1scFv-HSA-K500A ND ND ND scFv-HSA-D550N 7.5 ± 0.1 19.0 ± 0.2  2.5scFv-HSA-K573P 7.4 ± 0.1 0.8 ± 0.1 0.1 ^(a)Dilutions of HSA variantswere injected over immobilized shFcRn (~1500 RU). ^(b)The kinetic rateconstants were obtained using a simple first-order (1:1) bimolecularinteraction model. The kinetic values represent the average ofduplicates. ^(c)Not determined due to weak binding (ND).

In example 8 it was shown that the K500A variant did not significantlybind shFcRn, in Example 10 it was shown that the K573P and K573Avariants bind shFcRn stronger than HSA and in Example 11 it was shownthat the D550N variant binds FcRn weaker than HSA.

In the present example it is shown that these observed difference inbinding properties also are reflected in fusion polypeptides indifferent configurations: C-terminal fusions with a small moiety(HSA-FLAG), C-terminal fusions with a larger polypeptide (HSA-IL1RA);N-terminal fusions with polypeptide (scFv-HSA); N- and C-terminalfusions (scFv-HSA-FLAG and scFv-HSA-scFv-FLAG).

Example 17. Conjugation of Horseradish Peroxidase Protein to Albumin andthe K573P Variant

For conjugation analysis, commercially available recombinant albumin(Recombumin™) was used as a control molecule. For this example, a final200 mg/mL albumin K573P variant of the invention was purified from a fedbatch fermentation by means described in Material and Methods. A twostep purification was carried out;

The first step used a column (bed volume approximately 400 mL, bedheight 11 cm) packed with AlbuPure™ matrix (ProMetic). This wasequilibrated with 50 mM sodium acetate, pH 5.3 and loaded with neatculture supernatant, at approximately pH 5.5-6.5, to approximately 20mg/mL matrix. The column was then washed with approximately 5 columnvolumes each of 50 mM sodium acetate, pH 5.3, 50 mM sodium phosphate, pH6.0, 50 mM sodium phosphate, pH 7.0 and 50 mM ammonium acetate, pH 8.0,respectively. Bound protein was eluted using approximately two columnvolumes of 50 mM ammonium acetate, 10 mM octanoate, pH 7.0. The flowrate for the entire purification was 154 mL/min.

For the second step, the eluate from the first step was dilutedapproximately two fold with water to give a conductivity of 2.5±0.5mS/cm after adjustment to pH 5.5±0.3 with acetic acid. This was loadedonto a DEAE-Sepharose Fast Flow (GE Healthcare) column (bed volumeapproximately 400 mL, bed height 11 cm), equilibrated with 80 mM sodiumacetate, 5 mM octanoate, pH 5.5. Loading was approximately 30 mgprotein/mL matrix. The column was washed with approximately 5 columnvolumes of 80 mM sodium acetate, 5 mM octanoate, pH 5.5. Followed byapproximately 10 column volumes of 15.7 mM potassium tetraborate, pH9.2. The bound protein was eluted using two column volumes of 110 mMpotassium tetraborate, 200 mM sodium chloride, approximately pH 9.0. Theflow rate was 183 mL/min during the load and wash steps, and 169 mL/minduring the elution step.

The eluate was concentrated and diafiltered against 145 mM NaCl, using aPall Centramate Omega 10,000 Nominal MWCO membrane, to give a finalprotein concentration of approximately 200 mg/mL.

Both 200 mg/mL stock solutions of the rHA and K573P variant albumin werediluted down to 5 mg/mL, using phosphate buffer saline (PBS), pHadjusted to pH 6.5-6.7. This ensured a favourable pH environment for themaleimide reactive group of the EZ-Link® Maleimide Activated HorseradishPeroxidase (Thermo Scientific) to react with the free sulphydryl, toform a stable thioester bond. 2 mg of the EZ-Link® Maleimide ActivatedHorseradish Peroxidase (HRP) was mixed with either 1 mL of the 5 mg/MLrHA or K573P variant albumin. This mixture ensured an approximate 2 foldmolar excess of the albumin, or K573P variant albumin. This mixture wasminimally incubated at 4° C., for 24 hours. The reaction mixtures werethen checked for conjugation, using GP-HPLC.

To separate unconjugated species (rHA, or Albumin variant K573P andunreacted HRP) from the corresponding conjugated species the sampleswere first concentrated (Vivaspin20, 10,000 MWCO PES, Sartorius), andthen individually applied to a Tricorn Superdex™ 200, 10/300 GL column(GE Healthcare), run at a flow rate of 45 cm/hr in PBS. The elution peakwas fractionated and GP-HPLC analysed. Fractions containing theconjugated species were pooled, concentrated and diafiltered against 50mM NaCl and analysed by GP-HPLC to demonstrate (FIG. 24)

These samples were then assayed using the Biacore method describedherein (Table 21). This example demonstrates that the K573P maintainsits increased affinity for shFcRn compared the WT HSA.

Example 18. Conjugation of Fluorescein to Albumin and the K573P Variant

The two same albumin samples used in Example 17, were also the startmaterials for this example. I.e. Approximately 200 mg/mL rHA or theK573P albumin variant.

Fluorescein-5-Maleimide, Thermo Scientific (F5M) was dissolved indimethylformamide, to give a final concentration of 25 mg/mL. This wasthen further diluted into 18 mls of PBS, pH adjusted to approximately pH6.5. To this solution either 1 ml of 200 mg/mL rHA or 1 mL of 200 mg/mLK573P variant was added. This gave an approximate 20 fold final molarexcess of F5M. These samples were incubated and allowed to conjugateovernight at 4° C., in the dark, to allow the maleimide groups on theF5M to react with predominantly the free sulfhydryl, present in bothalbumin species.

Following overnight incubation aliquots of the reaction mixtures wereextensively diafiltered against 50 mM NaCl to remove unconjugated F5M,(Vivaspin20, 10,000 MWCO PES, Sartorius). Conjugation was confirmed byultraviolet visualization of conjugated Fluorescein::Albumins Followingstandard SDS-PAGE (FIG. 25).

These diafiltered samples were then assayed using the Biacore methoddescribed herein (Table 21). This example demonstrates that theconjugation of a small molecule to either rHA or a variant, e.g. K573Pdoes not affect the trend in binding affinities to shFcRn.

TABLE 21 Representative Biacore assay KD values of conjugated rHA or avariant (K573P) when binding to immobilized shFcRn. KD Analyte (μM)rHA::HRP 3.6 K573P::HRP 0.02 rHA::F5M 7.3 K573P::F5M 2.5

Example 19. Further Albumin Variants

The following variants were generated using the methods described inExample 1 E492T, N503D, E492T+N503D, K538H, E542D, D494N+E495Q+T496A,E495Q+T496A, N403K, K541A and K541N. SPR analysis was carried out asdescribed in Example 15 and the results presented in FIG. 26 and FIG.27.

FIGS. 30A and 30B shows the effect on shFcRn binding for the albuminvariants.Substitutions N503D, D494N+E495Q+T496A E492T+N503D, E495Q+T496A withinHSA had a negative impact on binding to shFcRn at pH5.5.

Example 20. Variants of Albumin at the C-Termini

The following variants were generated using the methods described inExample 1. Binding to the shFcRn was determined as described inMaterials and Methods and the results are presented in Table 22.

TABLE 22 Kinetics of the HSA C-terminal swapped variant interactionswith shFcRn. Albumin ka kd KD^(b) variant^(a) (10³/Ms) (10⁻³/s) (μM) HSA4.4 ± 0.0 24.0 ± 0.1  5.4 MacSA 3.1 ± 0.1 8.6 ± 0.1 2.7 HSA-MacC 4.1 ±0.1 5.6 ± 0.0 1.3 MouseSA^(c) 3.8 ± 0.0 3.1 ± 0.1 0.8 HSA-MouseC 3.7 ±0.1 1.3 ± 0.0 0.3 RabbitSA^(d) 1.9 ± 0.3 1.7 ± 0.1 0.9 HSA-RabC 3.5 ±0.0 1.6 ± 0.0 0.4 SheepSA ND ND ND HSA-SheepC 3.3 ± 0.0 2.1 ± 0.0 0.6^(a)Dilutions of HSA variants were injected over immobilized shFcRn(~1500 RU). ^(b)The kinetic rate constants were obtained using a simplefirst-order (1:1) bimolecular interaction model. ^(c)Data from Table 2^(d)Data from Table 3 Not determined due to weak binding (ND)

This example demonstrates that for all C-terminal swaps to human albumintested an increase in binding over the donor albumin was observed. Alldonor sequences contain the K573P substitution shown to significantlyincrease binding but less that the K573P alone (Table 20).

Example 21. Competitive Binding Analysis of Variant Albumin Fusions

Competitive binding studies, using variant albumin fusions and aselection of variant albumins prepared as described in Example 1, wereperformed as described in Example 4. Results are presented in FIGS.28-31.

The competitive binding hierarchy was identical for the variants fusionsof HSA-FLAG and, N+C-terminal scFv HSA-FLAG to the hierarchy of theindividual HSA variants (unfused and fused) affinity data. For the IL1Ravariants K573P, K573A, and the K500A were as predicted, however theD550N appears to inhibit more efficiently than the WT fusion.

Example 22. Further HSA Variants

The following variants were generated using methods described in Example1: HSA E492G+K573A, HSA E492G+N503K+K573A, HSA E492G+N503H+K573A, HSAE492G+K573P, HSA E492G+N503K+K573P, HSA E492G+N503H+K573P. SPR analysiswas performed as described in Materials and Methods. Results (FIG. 32)showed that all HSA variants bound more strongly to shFcRn compared towild type HSA at pH 5.5. No binding was observed at pH 7.4.

HSA E492G+K573A, HSA E492G+N503K+K573A, unlike HSA E492G+N503H+K573A,had marginally improved binding beyond that of HSA K573A. Thecombination variants containing K573P did not show improved binding overthe K573P single variant.

1. A Method for preparing a variant of albumin, fragments thereof orfusion polypeptide comprising said variant albumin or fragment thereof,comprising following steps: a. Providing a nucleic acid encoding aparent albumin having at least 80% sequence identity to SEQ ID NO: 2; b.Modifying the sequence of step a., to encode a variant albumin,fragments thereof or fusion polypeptide comprising said variant albuminor fragment thereof having one or more substitutions corresponding tothe substitutions in SEQ ID NO: 2 selected among:Q417A,C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,Y;H440A,C,D,E,F,G,I,K,L,M,N,P,Q,R,S,T,V,W,Y;A490C,D,E,F,G,H,I,K,L,M,N,P,R,S,T,V,W,YE492A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;V493A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T.W,Y;D494A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;E495A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;T496A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,V,W,Y; P499A,C,D,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y; K500A,C,D,E,F,G,H,I,L,M,N, Q,R,S,T,V,W,Y;E501A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; N503A,C,D,E,F,G,H,I,K,L,M,P,Q,R,S,T,V,W,Y; A504C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;E505A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;T506A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S, V,W,Y;H510A,C,D,F,G,I,K,L,M,N,P,Q,R,S,T,V,W,Y;H535A,C,D,F,G,I,K,L,M,N,P,Q,R,S,T,V,W,Y; K536A,C,D,E,F,G,H,I,L,M,N,P,Q,R,S,T,V,W,Y; P537A,C,D,E,F,G,H,I,K,L,M,N,Q,R,S,T,V,W,Y;K538A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y; T540A,C,D,E,F,G,H,I,L,M,N,P,Q,R,S,V,W,Y; K541A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y;E542A,C,D,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;D550A,C,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; K573A,C,D,E,F,G,H,I,L,M,N,P,Q,R,S,T,V,W,Y; K574A,C,D,E,F,G,H,I, L,M,N,P,Q,R,S,T,V,W,Y;Q580A,C,D,E,F,G,H,I, K, L,M,N,P,R,S,T,V,W,Y;A581C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y;A582C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; orG584A,C,D,E,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; c. Introducing the modifiedsequence of step b., in a suitable host cell; d. Growing the cells in asuitable growth medium under condition leading to expression of thevariant of albumin, fragments thereof or fusion polypeptide comprisingsaid variant albumin or fragment thereof; and e. Recovering the variantof albumin, fragments thereof or fusion polypeptide comprising saidvariant albumin or fragment thereof from the growth medium; wherein thevariant of albumin, fragments thereof or fusion polypeptide comprisingsaid variant albumin or fragment thereof, has an altered plasmahalf-life compared with the parent albumin, fragments thereof or fusionpolypeptide comprising said variant albumin or fragment thereof.